Protein chips, preparation and use thereof

ABSTRACT

The present invention relates to the field of medicine, in particular of research and diagnosis. It relates more particularly to a novel tool for detecting antibodies in a biological sample originating from a mammal. This tool, which is in the form of a protein chip, can be used in screening for new targets of interest involved in the occurrence of an autoimmune disease, in particular of a disease affecting the nervous system of a mammal, and also in the diagnosis or the monitoring of the progression of such an autoimmune disease. The invention also relates to a method for producing such a tool and also to kits comprising it and enabling its use.

The present invention relates to the field of medicine, in particular ofresearch and diagnosis. It relates more particularly to a novel tool fordetecting autoantibodies in a biological sample originating from amammal. This tool, which is in the form of a protein chip, can typicallybe used in screening for new targets of interest involved in theoccurrence of an autoimmune disease, in particular of a diseaseaffecting the nervous system of a mammal, and in the diagnosis or themonitoring of the progression of such an autoimmune disease. Theinvention also relates to a method for producing such a tool and also tokits comprising it and enabling its use.

PRIOR ART

The prior art provides several types of tests for detecting the presenceof a target molecule within a sample. Among these are radioimmunoassays(RIAs); immunofluorescence assays (IFAs); luminescence immunoassays(LIAs); EIA assays (enzyme immunoassays), in particular ELISA assays(enzyme-linked immunosorbent assays), i.e. enzymatic immunoassays on asolid support.

RIA (radioimmunoassay) makes it possible to measure antigenconcentrations in a sample using antibodies labelled with aradioelement. The assay of the radioelement measures a number ofdisintegrations per second. This technique is the reverse technique ofthe RBA (“radiobinding assay”) which makes it possible to quantify anantibody by means of the corresponding antigens. These techniques aresimple to carry out and inexpensive, but require the use of radioactivesubstances, often iodine isotopes bonded to tyrosine residues.Radioimmunoprecipitation assays (RIPAs) enable the precipitation of anantigen of interest present in a sample using a labelled specificantibody. This method can be used to isolate and concentrate aparticular protein within a sample comprising thousands of otherproteins. The technique requires the antibody to be coupled to a solidsubstrate.

Immunofluorescence assays (IFAs) use antibodies (or immunoglobulins) andalso fluorochromes (chemical substances capable of emitting fluorescentlight after excitation). The fluorochromes most commonly used arephycoerythrin (PE), fluorescein isothiocyanate (FITC), the Alexa Fluorrange, and green fluorescent protein (GFP). These assays have theadvantage of giving results very rapidly, or even immediately,dispensing with the long exposure times required for the technique withradioactivity. Fluorescence has the drawback of not being permanent, theintensity of the fluorescence decreasing over time until it becomesundetectable. In the context of luminescence immunoassays (LIAs), theenzymatic activity is measured by luminometry.

The ELISA (enzyme-linked immunosorbent assay) falls within the moregeneral context of EIAs (enzyme immunoassays) in which the assaying of areaction between antigen and antibody is coupled to a reaction catalyzedby an enzyme which releases a coloured component that is monitored byspectroscopy. The ELISA is a biochemical technique, mainly used inimmunology in order to detect the presence of an antibody or an antigenin a sample (e.g. Gotti et al. Muscle & Nerve, 20: 800-808, 1997;Franciotta et al., Clin Chem. 45: 400-5, 1999; Hewer et al., Clin ChimActa. 364:159-66, 2006). The technique uses one or two antibodies. Oneof them is specific to the antigen, while the other reacts with theimmune (antigen-antibody) complexes and is coupled to an enzyme. Thissecondary antibody, which is responsible for the name of the technique,can also cause the emission of a signal by a chromogenic or fluorogenicsubstrate.

The assays available and described above are not, however, sensitiveenough to detect very low concentrations of target molecules, typicallyof antibodies, in particular of autoantibodies and even moreparticularly of autoantibodies directed against membrane proteins and/ormembrane protein complexes.

Moreover, they must generally be used on small amounts of serum. Adilution thus often proves to be necessary in order to avoid anexcessive background noise. ELISA assays for example can require an atleast 50-fold dilution of the serum. Radioimmunoprecipitation assays(RIPAs) prefer the use of only a few microlitres (approximately 5microlitres) of test serum in order to avoid the use of large volumes ofantiserum generating unacceptable background noises (i.e. inappropriatevalues of the radioactivity of the negative controls) owing toexcessively large pellets. Even though some specific ELISA assays mayuse larger serum volumes (e.g. 50 to 100 microlitres), their use ishowever limited and their sensitivity is not increased. For example, acommercial ELISA kit from RSR Ltd for detecting anti-nicotinicacetylcholine receptor (AChR) antibodies associated with myastheniagravis allows the use of a combination of three anti-AChR monoclonalantibodies in a sandwich-type assay using 100 microlitres of serum.However, this technique does not increase the sensitivity of the assaycompared with the standard RIA assay. In addition, according to Irani etal. (Autoimmunity, February 2008; 41(1): 55-65), the ELISA technique,just like the “Western blotting” and “phage display” techniques, allowsthe identification of autoantibodies directed against non-conformationalepitopes, but not that of autoantibodies directed against functionalepitopes. The “Western blotting” technique, like immunohistochemistry,in particular involves the denaturation of the proteins of interest (H.Prüss et al: “Anti-NMDA-receptor encephalitis”, Der Nervenartz, vol. 81,no. 4, 31 Jan. 2010, pp. 396-408).

A certain number of recent studies (Vincent et al., Autoimmunechannelopathies and related neurological disorders. Neuron 200652:123-138, and Vernino et al., Autoimmune encephalopathies. Neurologist2007 13(3):140-7) show the presence and the involvement ofautoantibodies in pathological conditions affecting the nervous system.The notion that neurons can be the target of an autoimmune attack, inparticular in the central nervous system, is quite recent. The articleby Graus et al. (J. Antibodies and neuronal autoimmune disorders of theCNS. J. Neurol. 2010 257(4):509-17) suggests the detection ofautoantibodies for diagnosing paraneoplastic neurological syndromes. Thearticle by Irani et al. (Autoantibody-mediated disorders of the centralnervous system. Autoimmunity 2008 41(1):55-65) describes the existenceof autoantibodies directed against ion channels and surface receptorshaving pathogenic effects in myasthenia gravis and acquiredneuromyotonia. The pathogenicity of these antibodies is supported by thefact that encephalitis and paraneoplastic neurological syndromes (PNSs)respond positively to immunomodulatory treatments and to plasmapheresis.Furthermore, this positive therapeutic response correlates with adecrease in the titre of these autoantibodies assayed in thecerebrospinal fluid.

From a practical point of view, the regular description of newautoantibodies and the spectrum of associated syndromes are such thatneurologists still have trouble estimating which antibodies or which setof antibodies is of use for diagnosing a given syndrome.

The importance and the potential of such markers for diagnosis andtreatment are however in no doubt and require the development ofsystematic methods for detecting and characterizing these antibodies inorder to meet the need of very substantially improving correlationsbetween clinical syndrome, immunological signature and therapeuticresponse.

Among the pathological conditions affecting the nervous system(neurological disorders or disease) which do not at the current timebenefit from an adequate method of diagnosis are in particularencephalopathies, in particular those associated with neuronalexcitability disorders.

There is in fact at the current time no tool which is sufficientlysensitive and specific to be able to effectively detect theautoantibodies, often present in very small amounts, directed againstmembrane proteins or membrane protein complexes, in particular proteinsor complexes comprising functional epitopes, described as beinginvolved, or suspected of being involved, in the occurrence of theseencephalopathies in humans. Thus, for example, patent application WO2011/142900, which describes a method for the diagnosis of aneurodegenerative disease comprising the detection of autoantibodies andalso a chip fabricated using proteins, specifies however that theseproteins are produced in insect cells, and not, contrary to theinvention, in mammalian cells, before being purified. Such a productionmethod inevitably has an impact on the post-translational modificationsof the proteins thus produced and therefore the recognition of epitopesby possible autoantibodies. It is also impossible, using such a chip, toenable the coexpression of partner subunits (protein complex) at thesurface of said chip. The other types of tests for identifyingautoantibodies that are carried out at the current time generallyrequire, for their part, the sacrifice of animals, typically rodents(rats, mice), since they are all carried out on cells, in particular ontransfected cells, or on tissue sections (typically placed on glassslides).

SUMMARY OF THE INVENTION

The inventors presently describe, for the first time, a protein chip fordetecting antibodies (in particular identifying new antibodies),preferably autoantibodies, even more preferentially autoantibodiesdirected against membrane proteins and/or membrane protein complexes, ina biological sample. This chip comprises at least one solubilizedprotein of interest and is in the form of a support for detecting thecomplexes formed between antibodies, preferably autoantibodies, andepitopes specific for said protein and/or for said protein complex, itbeing possible for said specific epitopes to be sequential orconformational epitopes. In one preferred embodiment of the invention,said at least one solubilized protein of interest is a solubilizedmembrane protein of interest and the protein chip is a membrane proteinchip. In one even more preferred embodiment, the solubilized membraneprotein of interest is expressed in the cells of the nervous system ofan animal, preferably of a human being, and is involved or suspected ofbeing involved in the occurrence of an encephalopathy associated withneuronal excitation disorders.

A considerable advantage of the protein chip according to the inventionis that, owing to the preservation by the protein of its nativethree-dimensional structure, it makes it possible to detect bothsensitively and specifically any antibody, present in a biologicalsample, capable of binding to any one of the epitopes (sequential aswell as conformational) formed by the protein(s) of interest expressedon said chip. The membrane protein of interest may be alone (membraneprotein) or associated with one or more membrane or perimembraneauxiliary subunits (membrane protein complex). By virtue of itsspecificity, this chip allows, moreover, the use of large amounts ofbiological samples to be tested and does not impose, in the context ofits use, any step of dilution of said biological sample. It is thus nowpossible to detect the presence of antibodies even when theirconcentration in the sample to be tested is very low.

A particular subject of the invention relates to a protein chip for thesimple and effective identification of antibodies, preferably ofautoantibodies, in a biological sample, said chip comprising at leastone solubilized protein of interest, typically at least one solubilizedmembrane protein of interest, preferably chosen from an ion channelprotein, a transporter protein and a membrane receptor protein whichregulates the activity of an ion channel protein or of a transporterprotein, said protein being optionally associated with one or moreauxiliary subunits, and said chip being in the form of a support fordetection of the complexes formed between antibodies, preferablyautoantibodies, and (sequential and also conformational) epitopesspecific for said protein, optionally associated with its auxiliarysubunit(s). Preferably, said at least one protein or said at least onemembrane protein complex is expressed in the nervous system of a humanbeing and is preferably involved or suspected of being involved in theoccurrence of an encephalopathy associated with neuronal excitabilitydisorders.

Another subject of the invention relates to a protein chip comprisingseveral different proteins of interest, typically several differentmembrane proteins of interest, typically at least 50 or at least 100different proteins of interest, preferably between approximately 200 andapproximately 400 different proteins of interest, for exampleapproximately 250 or approximately 300 different proteins of interest,each protein of interest being optionally associated with one or moreauxiliary subunits.

Moreover, the invention relates to a method for preparing a chip asdescribed previously, and also the chip capable of being obtained bymeans of such a method, said method comprising the following steps ofcloning the cDNA(s) of interest, of expressing the protein(s) (typicallythe membrane protein(s)) encoded by said cDNA(s) in cells in culture, ofsolubilizing the proteins expressed using a non-denaturing detergentwhich enables the solubilization of said proteins while at the same timepreserving their native three-dimensional conformation and their abilityto bind to a support, and of depositing said solubilized proteins on asupport in order to obtain the chip of interest. Contrary to theinvention, patent application WO2004/108762 describes methods forpreparing solubilized thyroid membrane which all require a fractionationstep using salts in order to obtain a fraction enriched with proteins ofinterest, and also an ultracentrifugation step.

The invention also relates to the use of a chip according to theinvention for screening for antibodies of interest, preferably for anantibody involved in the occurrence of an autoimmune disease, or for thediagnosis and/or the monitoring of the progression of an autoimmunedisease.

Another subject of the invention relates, moreover, to a kit comprisinga chip as described previously and, optionally, one or more reagentspreferably chosen from a buffer for blocking the non-specific sites, abuffer allowing the association between antibodies and antigens (Ab/Ag),a washing buffer, a secondary antibody, one or more product(s) fordetecting said secondary antibody or antibodies, and instructions foruse.

DETAILED DESCRIPTION Products

The invention relates to a protein chip for detecting antibodies,typically autoantibodies, preferably autoantibodies directed againstmembrane proteins and/or membrane protein complexes, in a biologicalsample, and also the method for obtaining same and the uses thereof.More specifically, the invention relates to a protein chip for detectingautoantibodies, in particular identifying new autoantibodies, in abiological sample. The inventors describe, for the first time, in thecontext of the present invention, a chip in the form of a supportcomprising at least one solubilized protein of interest (typically asolubilized membrane protein of interest), preferably a protein havingretained its native three-dimensional structure (or conformation), andallowing the detection of the complexes formed between antibodies,typically autoantibodies, and epitopes (sequential or conformationalepitopes), typically conformational epitopes, specific for said proteinof interest.

The present invention allows, for the first time, the detection, whichis at the same time sensitive and specific, of the complexes formedbetween autoantibodies and epitopes (sequential and conformational) ofthe membrane protein of interest (optionally associated with itsauxiliary subunit(s)), preferably of a protein or of a protein complexinvolved or suspected of being involved in the occurrence of anencephalopathy associated with neuronal excitability disorders. Inaddition, this method makes it optional to dilute the biological sampletested.

The term “biological sample” encompasses any sample (fluid, tissue orcell) derived from an animal, typically from a mammal, from a lagomorphor from a rodent (rat, mouse, hamster, guinea pig), preferably from ahuman being. The biological sample is preferably a fluid, typically afluid containing autoantibodies produced by said animal. The biologicalsample can be obtained directly from said animal or could be derivedfrom a cell culture obtained from said animal. The biological samplesare preferably chosen, for example, from blood, serum, plasma,cerebrospinal fluid (CSF), inner ear endolymph, inner ear perilymph, anda subfraction or a product derived therefrom. A preferred biologicalsample is a cerebrospinal fluid sample. The biological sample may havebeen subjected to a treatment such as a dilution in an acceptablevehicle. However, the present invention advantageously makes it possibleto dispense with such a step.

The term “autoantibody” comprises any antibody and also any antibodyfragment or portions such as Fab, F(ab′)2, Fv and scFv which bind to anepitope of a self protein.

The term “protein” covers any amino acid sequence, or set of amino acidsequences, comprising at least one antigen comprising at least oneepitope. In the context of the present invention, the term “protein”typically covers a membrane protein or a membrane protein complex asdefined previously.

The term “solubilized protein” denotes a protein having been exposed toa non-denaturing detergent, i.e. a non-ionic detergent (also identifiedas “mild detergent”), allowing the solubilization of said proteins whileat the same time preserving their conformation (three-dimensionalstructure) and their ability to bind to a support. This mildsolubilization is the first step used for the purification of themembrane proteins and then subsequently their functional analysis.

The term “antigen” denotes a natural or synthetic macromoleculerecognized by antibodies or cells of the immune system and capable ofgenerating an immune response.

The term “epitope” (or “antigenic determinant”) denotes animmunoreactive sequence of amino acids, i.e., an amino acid sequencecapable, by virtue of its structure, of specifically interacting with aparticular antibody.

The same antigen may comprise several (identical or different) epitopesand thus induce varied immune responses. Sequential epitopes,corresponding to an amino acid sequence, and conformational epitopes,related to the authentic structure (spatial conformation or nativethree-dimensional conformation) of the protein, exist.

For the purposes of the invention, a protein can thus, for example,consist of a single-chain polypeptide; a set of polypeptides connectedby covalent or non-covalent bonds; a protein portion such as a subunit,a domain or a fragment; a modified protein, for example modified byglycosylation (glycoprotein) or by combination with a lipid(lipoprotein). The term “protein” also covers the variants, derivativesand analogues of a particular protein.

The term “variant” or “derivative” is intended to mean any amino acidsequence which has been spontaneously modified (natural variant) orintentionally modified (artificial variant) compared with the specificamino acid sequence encoding the particular protein, such that theprotein variant retains at least one of the endogenous functions of saidparticular protein, for example all of the endogenous functions of thisparticular protein (functionally equivalent protein). A variant can beobtained by addition or insertion, deletion and/or substitution of oneor more amino acid residue(s) within the specific amino acid sequenceencoding the particular protein.

The choice of the substitution amino acid can be decided on the basis ofsimilarity of polarity, charge, solubility, hydrophobicity,hydrophilicity and/or of amphiphatic nature guaranteeing thepreservation of a particular function (e.g. transport function ortransport activity modulating function). Negatively charged amino acidsinclude, for example, aspartic acid and glumatic acid, positivelycharged amino acids include, for example, lysine and arginine anduncharged amino acids having a similar hydrophilic nature include, forexample, leucine, isoleucine, valine, glycine, alanine, asparagine,glutamine, serine, threonine, phenylalanine and tyrosine. Conservativesubstitutions can also be made, for example according to Table 1 below.The amino acids identified in the same block in the second column andpreferably belonging to the same line in the third column can, forexample, substitute for one another.

TABLE 1 ALIPHATIC Non-polar G A P I L V Polar-Uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

The term “analogue” is intended to mean any mimetic which is a naturallyexisting or artificially produced chemical compound which has at leastone of the endogenous functions of the protein that it mimics, forexample all of the endogenous functions of the protein that it mimics(functionally equivalent protein).

Generally, an epitope consists of a sequence of at least 3 to 4 aminoacids, and more commonly of at least 5 to 6 amino acids. The epitopesmay also consist of a sequence of approximately 7 to 8 amino acids, ofeven of approximately 10 amino acids. In the case of a sequentialepitope, the amino acids are consecutive within the linear amino acidsequence of the protein. Conversely, as explained previously, the aminoacids of a conformational epitope are not necessarily all consecutivewithin the linear amino acid sequence of the protein, the conformationalepitope depending on the structure of the protein. In the context of thepresent invention, a particular epitope of a given protein denotes a(linear or conformational) particular amino acid sequence and also theimmunological equivalents of the latter.

In one particular embodiment, the invention relates to a protein chipfor detecting antibodies, typically autoantibodies, in a biologicalsample, said chip comprising at least one solubilized protein ofinterest (preferably a protein having retained its three-dimensionalstructure), typically at least one solubilized membrane protein ofinterest chosen, for example, from an ion channel protein, a transporterprotein, and a membrane receptor protein which regulates the activity ofan ion channel protein or of a transporter protein, said protein beingoptionally, when it corresponds to a main subunit, associated with oneor more auxiliary subunits, and said chip being in the form of a supportfor detection of the complexes formed between antibodies and epitopesspecific for said protein, the latter being optionally associated withits auxiliary subunit(s).

The term “ion channel protein” denotes a membrane protein which allowsthe passive passage of one or more ions. Ion channel proteins capable ofbeing bound to the surface of the protein chip according to theinvention can be chosen, for example, from ion channels and channelreceptors.

Examples of ion channels are potassium channels (intracellularcalcium-activated potassium channel, inwardly rectifying potassiumchannel, two-P-domain potassium channel, etc.), calcium channels, sodiumchannels and chloride channels. The ion channel may be a voltage gatedchannel.

Examples of channel receptors are the acetylcholine receptor, theglutamate receptor, the γ-aminobutyric acid (GABA) receptor and theadenosine-5′-triphosphate (ATP) receptor.

The term “protein transporter” denotes a protein intrinsic to the lipidcell membrane which allows the selective passage of water, of ionsand/or of metabolites. Transporter proteins capable of being bound tothe surface of the protein chip according to the invention can be chosenfrom, for example, aquaporins, connexins, Na+/K+ ATPases (or Na+/K+pumps), H+/K+ ATPases (or H+/K+ pumps), Na+/H+ exchangers, Na+/Ca++exchangers, and Na—K—Cl cotransporters.

The expression “membrane receptor protein which regulates the activityof an ion channel protein or of a transporter protein” or “regulatorymembrane receptor protein” denotes any membrane protein, optionallyassociated with an ion channel protein and/or with a transporterprotein, which regulates at least one activity of said proteins.Regulatory proteins of membrane receptor type which are capable of beingbound to the surface of the protein chip according to the invention canbe chosen, for example, from seven-transmembrane domains Gprotein-coupled receptors and receptors of tyrosine kinase type.

As indicated above, said at least one protein of interest deposited onthe support typically corresponds to a single-stranded protein (peptide)in its three-dimensional configuration, to the main subunit of amultistrand protein (polypeptide) in its natural configuration, i.e.three-dimensional configuration, or else to one of the main subunits ofsuch a multistrand protein. This protein of interest may, however,correspond to or be associated with one or more auxiliary subunits of aset of functionally associated proteins (protein complex).

The term “auxiliary subunit” denotes the secondary parts of a protein ora protein complex, i.e. the parts other than the main subunit(s). Theauxiliary subunit may be a membrane or perimembrane subunit. Theauxiliary subunits of a protein often make it possible to regulate theactivity (i.e. at least one of the functions) or a property (i.e. afunctional characteristic) of said protein. In the case of an ionchannel or of a transporter protein, for example, the main subunitgenerally allows the passage of ions or of metabolites, while theauxiliary subunits regulate this passage. Chaperone proteins andionotropic receptors (“ionophores”) are examples of auxiliary subunitsof a protein complex.

Preferably, the protein of interest is expressed in the cells of thenervous system of an animal, typically of a mammal, of a rodent (rat,mouse, hamster, guinea pig) or of a lagomorph, preferably of a humanbeing. The protein of interest is thus preferably a human protein. Itmay also be a protein originating from an animal other than a humanbeing, such as those previously identified. Preferably, such an animalprotein has a sequence sufficiently close to that of the correspondinghuman isoform to enable the recognition, by the antibodies of the immunesystem of a human being, of at least one conformational epitope of saidanimal protein. Typically, the animal protein of interest exhibits ahomology between its linear sequence and that of the human isoform of atleast 50%, preferably of at least 75%, even more preferentially of atleast 95%.

The protein of interest is typically a human membrane protein selectedfrom a pentameric receptor (or “cys-loop” receptor), an ATP receptor, anionotropic glutamate receptor, a voltage gated channel, for example avoltage gated calcium, sodium or potassium channel, an intracellularcalcium-activated potassium channel, an inwardly rectifying potassiumchannel, a two-P-domain potassium channel, a cyclicnucleotide-activatable ion channel, a TRP (Transient Receptor Potential)ion channel, a chloride channel, an acid-sensitive cation channel, asodium channel auxiliary subunit, and a calcium channel auxiliarysubunit.

As previously indicated, the protein of interest may also be a rodentprotein, typically a mouse or rat protein. Membrane proteins of interestoriginating from rodents can, for example, be chosen from a rat sodiumchannel, a rat calcium channel and a rat glutamate channel.

In the context of the present invention, the protein of interest,typically the membrane protein of interest, is preferably associated (orsuspected of being associated) with a human pathological conditionresponsible for the appearance of autoantibodies.

In one particular embodiment of the invention, the protein of interest,typically the membrane protein of interest, is advantageously associated(or suspected of being associated) with an autoimmune (or presumedautoimmune) disease or pathological condition affecting the nervoussystem (neurological disease).

Examples of autoimmune pathological conditions affecting the nervoussystem comprise encephalopathies associated with neuronal excitabilitydisorders, typically with neuronal receptor excitability disorders, andinclude, without being limited thereto, acquired epileptic syndromes,encephalitis, paraneoplastic neurological syndromes (PNSs), andnon-paraneoplastic neurological syndromes with autoantibodies. Examplesof convulsive or non-convulsive antoimmune (or presumed autoimmune)encephalopathies, associated with neuronal excitability disorders, arelimbic encephalitis (which involves autoantibodies directed againstproteins associated with voltage gated potassium channels orautoantibodies directly directed against these channels), Rasmussenencephalitis (associated with antibodies directed against glutamatereceptors and acetylcholine receptors), post-infectious encephalitis,anti-NMDA receptor (anti-NMDAR) antibody encephalitis, Morvan'ssyndrome, Devic's disease or Devic's neuromyelitis optica (whichinvolves autoantibodies directed against aquaporin 4), opsoclonusmyoclonus syndromes in children (of post-infectious or paraneoplasticorigin), encephalomyelitis, acquired epileptic syndromes, andparaneoplastic neurological syndromes (PNSs). Preferentially, theencephalopathy is an attack on the central nervous system which may beassociated with neuronal excitability disorders of (demonstrated orsuspected) autoimmune origin and of post-infectious or paraneoplasticorigin.

In another particular embodiment of the invention, the protein ofinterest, typically the membrane protein of interest, is a proteinassociated (or suspected of being associated) with a peripheralneurological syndrome (possibly associated with an encephalopathy) of(demonstrated or suspected) autoimmune origin, and typically ofpost-infectious or paraneoplastic origin. The peripheral neurologicalsyndromes may be responsible for muscle paralysis of pre- orpost-synaptic origin. Isaac's syndrome is an example of peripheralneurological syndrome.

In another particular embodiment of the invention, the protein ofinterest, typically the membrane protein of interest, is a proteinassociated (or suspected of being associated) with a neurosensorydisease (possibly associated with sensory receptor excitabilitydisorders), for example with an inner ear disease, such as Menière'sdisease or acquired or presumed congenital deafness.

A particular chip according to the invention comprises at least onemembrane protein of interest, typically several membrane proteins ofinterest, said protein being encoded by a human cDNA sequence chosenfrom the nucleotide sequences identified in table 2 below orcorresponding to an amino acid sequence chosen from the amino acidsequences also identified in Table 2 below, preferably from the aminoacid sequences SEQ ID NO:1 to SEQ ID NO:311.

TABLE 2 Amplified Protein Seq Ref (isoforms) Gene Complete name of thegene Nucleotide Seq Ref (isoforms) gene size (bp) and associated SEQ IDNOs “Cys-loop” receptors HTR3A serotonin 5-HT3 receptor A NM_000869(Hs), NM_213621 1537 NP_000860 (Hs) (SEQ ID NO: 1), subunit (Hs),NM_013561 (Mm), NP_998786 (Hs) (SEQ ID NO: 2), NM_001099644 (Mm),NP_038589 (Mm), NP_001093114 NM_024394 (Rn) (Mm), NP_077370 (Rn) HTR3Bserotonin 5-HT3 receptor B NM_006028 (Hs), NM_020274 1333 NP_006019 (Hs)(SEQ ID NO: 3), subunit (Mm), NM_022189 (Rn) NP_064670 (Mm), NP_071525(Rn) HTR3C serotonin 5-HT3 receptor C NM_130770 1348 NP_570126 (Hs) (SEQID NO: 4) subunit HTR3D serotonin 5-HT3 receptor D NM_182537 844NP_872343 (Hs) (SEQ ID NO: 5) subunit HTR3E serotonin 5-HT3 receptor ENM_182589 1420 NP_872395 (Hs) (SEQ ID NO: 6) subunit CHRNA1 nicotinicacetylcholine receptor NM_001039523 (Hs), 1450 NP_001034612 (Hs) (SEQ IDNO: 7), α1 subunit NM_007389 (Mm), NM_024485 NP_031415 (Mm), NP_077811(Rn) (Rn) CHRNA2 nicotinic acetylcholine receptor NM_000742 (Hs),NM_144803 1591 NP_000733 (Hs) (SEQ ID NO: 8), α2 subunit (Mm), NM_133420(Rn) NP_659052 (Mm), NP_596911 (Rn) CHRNA3 nicotinic acetylcholinereceptor NM_000743 (Hs), NM_145129 1519 NP_000734 (Hs) (SEQ ID NO: 9),α3 subunit (Mm), NM_052805 (Rn) NP_660111 (Mm), NP_434692 (Rn) CHRNA4nicotinic acetylcholine receptor NM_000744 (Hs), NM_015730 1885NP_000735 (Hs) (SEQ ID NO: 10), α4 subunit (Mm), NM_024354 (Rn)NP_056545 (Mm), NP_077330 (Rn) CHRNA5 nicotinic acetylcholine receptorNM_000745 (Hs), NM_176844 1407 NP_000736 (Hs) (SEQ ID NO: 11), α5subunit (Mm), NM_017078 (Rn) NP_789814 (Mm), NP_058774 (Rn) CHRNA6nicotinic acetylcholine receptor NM_004198 (Hs), NM_021369 1489NP_004189 (Hs) (SEQ ID NO: 12), α6 subunit (Mm), NM_057184 (Rn)NP_067344 (Mm), NP_476532 (Rn) CHRNA7 nicotinic acetylcholine receptorNM_000746 (Hs), NM_007390 1513 NP_000737 (Hs) (SEQ ID NO: 13), α7subunit (Mm), NM_012832 (Rn) NP_031416 (Mm), NP_036964 (Rn) CHRNA9nicotinic acetylcholine receptor NM_017581 (Hs), 1444 NP_060051 (Hs)(SEQ ID NO: 14), α9 subunit NM_001081104 (Mm), NP_001074573 (Mm),NP_075219 NM_022930 (Rn) (Rn) CHRNA10 nicotinic acetylcholine receptorNM_020402 (Hs), 1354 NP_065135 (Hs) (SEQ ID NO: 15), α10 subunitNM_001081424 (Mm), NP_001074893 (Mm), NP_072161 NM_022639 (Rn) (Rn)CHRNB1 nicotinic acetylcholine receptor NM_000747 (Hs), NM_009601 1510NP_000738 (Hs) (SEQ ID NO: 16), β1 subunit (Mm), NM_012528 (Rn)NP_033731 (Mm), NP_036660 (Rn) CHRNB2 nicotinic acetylcholine receptorNM_000748 (Hs), NM_009602 1510 NP_000739 (Hs) (SEQ ID NO: 17), β2subunit (Mm), NM_019297 (Rn) NP_033732 (Mm), NP_062170 (Rn) CHRNB3nicotinic acetylcholine receptor NM_000749 (Hs), NM_173212 1381NP_000740 (Hs) (SEQ ID NO: 18), β3 subunit (Mm), NM_133597 (Rn)NP_775304 (Mm), NP_598281 (Rn) CHRNB4 nicotinic acetylcholine receptorNM_000750 (Hs), NM_148944 1501 NP_000741 (Hs) (SEQ ID NO: 19), β4subunit (Mm), NM_052806 (Rn) NP_683746 (Mm), NP_434693 (Rn) CHRNDnicotinic acetylcholine receptor NM_000751 (Hs), NM_021600 1555NP_000742 (Hs) (SEQ ID NO: 20), δ subunit (Mm), NM_019298 (Rn) NP_067611(Mm), NP_062171 (Rn) CHRNE nicotinic acetylcholine receptor NM_000080(Hs), NM_009603 1483 NP_000071 (Hs) (SEQ ID NO: 21), ε subunit (Mm),NM_017194 (Rn) NP_033733 (Mm), NP_058890 (Rn) CHRNG nicotinicacetylcholine receptor NM_005199 (Hs), NM_009604 1558 NP_005190 (Hs)(SEQ ID NO: 22), γ subunit (Mm), NM_019145 (Rn) NP_033734 (Mm),NP_062018 (Rn) GABRA1 GABA receptor α1 subunit NM_000806 (Hs), NM_0102501375 NP_000797 (Hs) (SEQ ID NO: 23), (Mm), NM_183326 (Rn) NP_034380(Mm), NP_899155 (Rn) GABRA2 GABA receptor α2 subunit NM_000807 (Hs),NM_008066 1360 NP_000798 (Hs) (SEQ ID NO: 24), (Mm) NP_032092 (Mm)GABRA3 GABA receptor α3 subunit NM_000808 (Hs), NM_008067 1483 NP_000799(Hs) (SEQ ID NO: 25), (Mm), NM_017069 (Rn) NP_032093 (Mm), NP_058765(Rn) GABRA4 GABA receptor α4 subunit NM_000809 (Hs), NM_010251 1666NP_000800 (Hs) (SEQ ID NO: 26), (Mm), NM_080587 (Rn) NP_034381 (Mm),NP_542154 (Rn) GABRA5 GABA receptor α5 subunit NM_000810 (Hs), NM_1769421390 NP_000801 (Hs) (SEQ ID NO: 27), (Mm), NM_017295 (Rn) NP_795916(Mm), NP_058991 (Rn) GABRA6 GABA receptor α6 subunit NM_000811 (Hs),1363 NP_000802 (Hs) (SEQ ID NO: 28), NM_001099641 (Mm), NP_001093111(Mm), NP_068613 NM_021841 (Rn) (Rn) GABRB1 GABA receptor β1 subunitNM_000812 (Hs), NM_008069 1429 NP_000803 (Hs) (SEQ ID NO: 29), (Mm),NM_012956 (Rn) NP_032095 (Mm), NP_037088 (Rn) GABRB2 GABA receptor β2subunit NM_021911 (Hs), NM_000813 1543 NP_068711 (Hs) (SEQ ID NO: 30),(Hs), NM_008070 (Mm), NP_000804 (Hs) (SEQ ID NO: 31), NM_012957 (Rn)NP_032096 (Mm), NP_037089 (Rn) GABRB3 GABA receptor β3 subunit NM_000814(Hs), NM_008071 1426 NP_000805 (Hs) (SEQ ID NO: 32), (Mm), NM_017065(Rn) NP_032097 (Mm), NP_058761 (Rn) GABRD GABA receptor δ subunitNM_000815 (Hs), NM_008072 1360 NP_000806 (Hs) (SEQ ID NO: 33), (Mm),NM_017289 (Rn) NP_032098 (Mm), NP_058985 (Rn) GABRE* GABA receptor εsubunit * NM_004961 (Hs), NM_017369 1525 NP_004952 (Hs) (SEQ ID NO: 34),(Mm), NM_023091 (Rn) NP_059065 (Mm), NP_075579 (Rn) GABRG1 GABA receptorγ1 subunit NM_173536 (Hs), NM_010252 1399 NP_775807 (Hs) (SEQ ID NO:35), (Mm), NM_080586 (Rn) NP_034382 (Mm), NP_542153 (Rn) GABRG2 GABAreceptor γ2 subunit NM_198904 (Hs), NM_008073 1432 NP_944494 (Hs) (SEQID NO: 36), (Mm), NM_183327 (Rn) NP_032099 (Mm), NP_899156 (Rn) GABRG3GABA receptor γ3 subunit NM_033223 (Hs), NM_008074 1405 NP_150092 (Hs)(SEQ ID NO: 37), (Mm), NM_024370 (Rn) NP_032100 (Mm), NP_077346 (Rn)GABRP GABA receptor π subunit NM_014211 (Hs), NM_146017 1183 NP_055026(Hs) (SEQ ID NO: 38), (Mm), NM_031029 (Rn) NP_666129 (Mm), NP_112291(Rn) GABRR1 GABA receptor ρ1 subunit NM_002042 (Hs), NM_008075 1322NP_002033 (Hs) (SEQ ID NO: 39), (Mm), NM_017291 (Rn) NP_032101 (Mm),NP_058987 (Rn) GABRR2 GABA receptor ρ2 subunit NM_002043 (Hs), NM_0080761474 NP_002034 (Hs) (SEQ ID NO: 40), (Mm), NM_017292 (Rn) NP_032102(Mm), NP_058988 (Rn) GABRR3 GABA receptor ρ3 subunit NM_001105580 (Hs),1405 NP_001099050 (Hs) (SEQ ID NO: 41), NM_001081190 (Mm), NP_001074659(Mm), NP_620252 NM_138897 (Rn) (Rn) GABRQ GABA receptor θ subunitNM_018558 (Hs), NM_020488 1183 NP_061028 (Hs) (SEQ ID NO: 42), (Mm),NM_031733 (Rn) NP_065234 (Mm), NP_113921 (Rn) GLRA1 glycine receptor α1subunit NM_000171 (Hs), NM_020492 1354 NP_000162 (Hs) (SEQ ID NO: 43),(Mm), NM_013133 (Rn) NP_065238 (Mm), NP_037265 (Rn) GLRA2 glycinereceptor α2 subunit NM_002063 (Hs), NM_183427 1363 NP_002054 (Hs) (SEQID NO: 44), (Mm), NM_012568 (Rn) NP_906272 (Mm), NP_036700 (Rn) GLRA3glycine receptor α3 subunit NM_006529 (Hs), NM_080438 1396 NP_006520(Hs) (SEQ ID NO: 45), (Mm), NM_053724 (Rn) NP_536686 (Mm), NP_446176(Rn) GLRA4 glycine receptor α4 subunit NM_001024452.2 (Hs), 1135NP_001019623 (Hs) (SEQ ID NO: 46), NM_010297 (Mm) NP_034427 (Mm),XP_346351.2 (Rn) GLRB glycine receptor β subunit NM_000824 (Hs),NM_010298 1498 NP_000815 (Hs) (SEQ ID NO: 47), (Mm), NM_053296 (Rn)NP_034428 (Mm), NP_445748 (Rn) ATP receptors P2RX1 ATP receptor P2X1subunit NM_002558 (Hs), NM_008771 1201 NP_002549 (Hs) (SEQ ID NO: 48),(Mm), NM_012997 (Rn), NP_032797 (Mm), NP_037129 (Rn), AF231010 (Rn)Q9JIF8 (Rn) P2RX2 ATP receptor P2X2 subunit AF190824 (Hs), AF190823(Hs), 1417 AAF19173 (Hs) (SEQ ID NO: 49), Isoform A AF190825 (Hs),NM_170682 NP_733782 (Hs) (SEQ ID NO: 50), (Hs), NM_153400 (Mm), AAF19171(Hs) (SEQ ID NO: 51), NM_053656 (Rn) AAF19172 (Hs) (SEQ ID NO: 52),NP_700449 (Mm), NP_446108 (Rn) P2RX3 ATP receptor P2X3 subunit NM_002559(Hs), NM_145526 1198 NP_002550 (Hs) (SEQ ID NO: 53), (Mm), NM_031075(Rn) NP_663501 (Mm), NP_112337 (Rn) P2RX4 ATP receptor P2X4 subunitNM_002560 (Hs), NM_011026 1168 NP_002551 (Hs) (SEQ ID NO: 54), (Mm),NM_031594 (Rn) NP_035156 (Mm), NP_113782 (Rn) P2RX5 ATP receptor P2X5subunit NM_002561 (Hs), NM_033321 1270 NP_002552 (Hs) (SEQ ID NO: 55),isoform A (Mm), NM_080780 (Rn) NP_201578 (Mm), NP_542958 (Rn) P2RXL1 ATPreceptor P2X6 subunit (aka NM_005446 (Hs), NM_011028 1330 NP_005437 (Hs)(SEQ ID NO: 56), P2XM, and P2X-like 1) (Mm), NM_012721 (Rn) NP_035158(Mm), NP_036853 (Rn) P2RX7 ATP receptor P2X7 subunit NM_002562 (Hs),NM_011027 1792 NP_002553 (Hs) (SEQ ID NO: 57), (Mm), FJ436444 (Mm),NP_035157 (Mm), ACR61395 (Mm), NM_019256 (Rn), FJ436445 ACR61396 (Rn),NP_062129 (Rn) (Rn) Ionotropic glutamate receptors Orphan glutamatereceptor δ1 subunit NM_017551 (Hs), NM_008166 3031 NP_060021 (Hs) (SEQID NO: 58), GRID1 (Mm), NM_024378 (Rn) NP_032192 (Mm), NP_077354 (Rn)Orphan glutamate receptor δ2 subunit NM_001510 (Hs), NM_008167 3025NP_001501 (Hs) (SEQ ID NO: 59), GRID2 (Mm), NM_024379 (Rn) NP_032193(Mm), NP_077355 (Rn) Kainate glutamate receptor KA1 subunit NM_014619(Hs), NM_175481 1875 NP_055434 (Hs) (SEQ ID NO: 60), GRIK4 (aka EAA1)(Mm), NM_012572 (Rn) NP_780690 (Mm), NP_036704 (Rn) Kainate glutamatereceptor KA2 subunit NM_002088 (Hs), NM_008168 2947 NP_002079 (Hs) (SEQID NO: 61), GRIK5 (aka EAA2) (Mm), NM_031508 (Rn) NP_032194 (Mm),NP_113696 (Rn) NMDA glutamate receptor NMDAR1 NM_007327 (Hs), NM_0081692665 NP_015566 (Hs) (SEQ ID NO: 62), GRIN1 subunit (Mm), NM_017010 (Rn)NP_032195 (Mm), NP_058706 (Rn) NMDA glutamate receptor NMDAR2A NM_000833(Hs), NM_008170 4396 NP_000824 (Hs) (SEQ ID NO: 63), GRIN2A subunit(Mm), NM_012573 (Rn) NP_032196 (Mm), NP_036705 (Rn) NMDA glutamatereceptor NMDAR2B NM_000834 (Hs), NM_008171 4459 NP_000825 (Hs) (SEQ IDNO: 64), GRIN2B subunit (Mm), NM_012574 (Rn) NP_032197 (Mm), NP_036706(Rn) NMDA glutamate receptor NMDAR2C NM_000835 (Hs), NM_010350 3706NP_000826 (Hs) (SEQ ID NO: 65), GRIN2C subunit (Mm), NM_012575 (Rn)NP_034480 (Mm), NP_036707 (Rn) NMDA glutamate receptor NMDAR2D NM_000836(Hs), NM_008172 4015 NP_000827 (Hs) (SEQ ID NO: 66), GRIN2D subunit(Mm), NM_022797 (Rn) NP_032198 (Mm), NP_073634 (Rn) NMDA glutamatereceptor NMDAR3A NM_133445 (Hs), 3352 NP_597702 (Hs) (SEQ ID NO: 67),GRIN3A subunit NM_001033351 (Mm) NP_001028523 (Mm) NMDA glutamatereceptor NMDAR3B NM_138690 (Hs), NM_130455 3133 NP_619635 (Hs) (SEQ IDNO: 68), GRIN3B subunit (aka χ-2) (Mm), NM_133308 (Rn) NP_569722 (Mm),NP_579842 (Rn) AMPA glutamate receptor GLUR1 NM_000827 (Hs), 2725NP_000818 (Hs) (SEQ ID NO: 69), GRIA1 subunit (aka KR4, HBGR1,NM_001114183 (Hs), NP_001107655 (Hs) (SEQ ID NO: 70), GluHI) NM_008165(Mm), NP_001106796 (Mm), NP_032191 NM_001113325 (Mm), (Mm), NP_113796(Rn) NM_031608 (Rn) AMPA glutamate receptor GLUR2 NM_001083619 (Hs),2656 NP_000817 (Hs) (SEQ ID NO: 71), GRIA2 subunit NM_000826 (Hs),NM_013540 NP_001077088 (Hs) (SEQ ID NO: 72), (Mm), NM_001083806 (Mm),NP_001077275 (Mm), NP_001034284 NM_001039195 (Mm), (Mm), NP_038568 (Mm),NP_058957 NM_017261 (Rn), (Rn), NP_001077280 (Rn) NM_001083811 (Rn) AMPAglutamate receptor GLUR3 NM_007325 (Hs), NM_000828 2686 NP_015564 (Hs)(SEQ ID NO: 73), GRIA3 subunit (aka GLURC) (Hs), NM_016886 (Mm),NP_000819 (Hs) (SEQ ID NO: 74), NM_001112742 (Rn), NP_058582 (Mm),NP_116785 (Rn), NM_032990 (Rn) NP_001106213 (Rn) AMPA glutamate receptorGluR4 NM_001077243 (Hs), 2713 NP_000820 (Hs) (SEQ ID NO: 75), GRIA4subunit NM_000829 (Hs), NP_001070711 (Hs) (SEQ ID NO: 76), NM_001077243(Hs), NP_000820 (Hs), NP_001070711 (Hs), NM_000829 (Hs), NP_062665 (Mm),NP_001106651 NM_001113180 (Mm), (Mm), NP_058959 (Rn), NM_019691 (Mm),NP_001106655 (Rn) NM_001113184 (Rn), NM_017263 (Rn) Kainate glutamatereceptor GLUR5 NM_175611 (Hs), NM_010348 2761 NP_783300 (Hs) (SEQ ID NO:77), GRIK1 subunit (aka EEA3) (Mm), NM_017241 (Rn) NP_034478 (Mm),NP_058937 (Rn) Kainate glutamate receptor GLUR6 NM_021956 (Hs),NM_010349 2731 NP_068775 (Hs) (SEQ ID NO: 78), GRIK2 subunit (aka EAA4aka β2) (Mm), NM_019309 (Rn) NP_034479 (Mm), NP_062182 (Rn) Kainateglutamate receptor GLUR7 NM_000831 (Hs), 2764 NP_000822 (Hs) (SEQ ID NO:79), GRIK3 subunit (aka EAA5) NM_001081097 (Mm), NP_001074566 (Mm),NP_001106187 NM_001112716 (Rn) (Rn) Voltage gated sodium channels SCN1Avoltage gated sodium channel NM_006920 (Hs), NM_018733 5998 NP_008851(Hs) (SEQ ID NO: 80), type I a subunit (Mm), NM_030875 (Rn) NP_061203(Mm) (SEQ ID NO: 81), NP_110502 (Rn) (SEQ ID NO: 82) SCN2A voltage gatedsodium channel NM_001040143 (Hs), 6019 NP_001035232 (Hs) (SEQ ID NO:83), type II a subunit NM_001040142 (Hs), NP_001035233 (Hs) (SEQ ID NO:84), NM_001099298 (Mm), NP_001092768 (Mm) (SEQ ID NO: NM_012647 (Rn)85), NP_036779 (Rn) (SEQ ID NO: 86) SCN3A voltage gated sodium channelNM_006922 (Hs), NM_018732 6004 NP_008853 (Hs) (SEQ ID NO: 87), type IIIa subunit (Mm), NM_013119 (Rn) NP_061202 (Mm) (SEQ ID NO: 88), NP_037251(Rn) (SEQ ID NO: 89) SCN4A voltage gated sodium channel NM_000334 (Hs),NM_133199 5512 NP_000325 (Hs) (SEQ ID NO: 90), type IV a subunit (Mm),NM_013178 (Rn) NP_573462 (Mm) (SEQ ID NO: 91), NP_037310 (Rn) (SEQ IDNO: 92) SCN5A voltage gated sodium channel NM_001099404 (Hs), 6055NP_001092874 (Hs) (SEQ ID NO: 93), type V a subunit NM_000335 (Hs),NM_198056 NP_001092875 (Hs) (SEQ ID NO: 94), (Hs), NM_001099405 (Hs),NP000326 (Hs) (SEQ ID NO: 95), NM_021544 (Mm), NM_013125 NP_932173 (Hs)(SEQ ID NO: 96), (Rn) NP_067519 (Mm) (SEQ ID NO: 97), NP_037257 (Rn)(SEQ ID NO: 98) SCN8A voltage gated sodium channel NM_014191 (Hs), 5944NP_055006 (Hs) (SEQ ID NO: 99), type VIII a subunit NM_001077499 (Mm),NP_001070967 (Mm) (SEQ ID NO: NM_019266 (Rn) 100), NP_062139 (Rn) (SEQID NO: 101) SCN9A voltage gated sodium channel NM_002977 (Hs), NM_0188525935 NP_002968 (Hs) (SEQ ID NO: 102), type IX a subunit (Mm), NM_133289(Rn) NP_061340 (Mm) (SEQ ID NO: 103), NP_579823 (Rn) (SEQ ID NO: 104)SCN10A voltage gated sodium channel NM_006514 (Hs), NM_009134 5872NP_006505 (Hs) (SEQ ID NO: 105), type X a subunit (Mm), NM_017247 (Rn)NP_033160 (Mm) (SEQ ID NO: 106), NP_058943 (Rn) (SEQ ID NO: 107) SCN11Avoltage gated sodium channel NM_014139 (Hs), NM_011887 5377 NP_054858(Hs) (SEQ ID NO: 108), type XI a subunit (Mm), NM_019265 (Rn) NP_036017(Mm) (SEQ ID NO: 109), NP_062138 (Rn) (SEQ ID NO: 110) Voltage gatedcalcium channels CACNA1S voltage gated calcium channel NM_000069 (Hs),5626 NP_000060 (Hs) (SEQ ID NO: 111), L-type a1S subunit NM_001081023(Mm), NP_001074492 (Mm) (SEQ ID NO: NM_053873 (Rn) 112), NP_446325 (Rn)(SEQ ID NO: 113) CACNA1C voltage gated calcium channel NM_000719 (Hs),NM_009781 6418 NP_000710 (Hs) (SEQ ID NO: 114), L-type a1C subunit (Mm),NM_012517 (Rn) NP_033911 (Mm) (SEQ ID NO: 115), NP_036649 (Rn) (SEQ IDNO: 116) CACNA1D voltage gated calcium channel NM_000720 (Hs), NM_0289816550 NP_000711 (Hs) (SEQ ID NO: 117), L-type a1D subunit (Mm), NM_017298(Rn) NP_083257 (Mm) (SEQ ID NO: 118), NP_058994 (Rn) (SEQ ID NO: 119)CACNA1F voltage gated calcium channel NM_005183 (Hs), NM_019582 5938NP_005174 (Hs) (SEQ ID NO: 120), L-type a1F subunit (Mm), NM_053701 (Rn)NP_062528 (Mm) (SEQ ID NO: 121), NP_446153 (Rn) (SEQ ID NO: 122) CACNA1Avoltage gated calcium channel NM_023035 (Hs), 6802 NP_075461 (Hs) (SEQID NO: 123), P/Q-type a1A subunit NM_000068.3 (Hs), NP_000059 (Hs) (SEQID NO: 124), NM_007578 (Mm), NM_012918 NP_031604 (Mm) (SEQ ID NO: 125),(Rn) NP_037050 (Rn) (SEQ ID NO: 126) CACNA1B voltage gated calciumchannel NM_000718 (Hs), 7021 NP_000709 (Hs) (SEQ ID NO: 127), N-type a1Bsubunit NM_001042528 (Mm), NP_001035993 (Mm) (SEQ ID NO: NM_147141 (Rn)128), NP_671482 (Rn) (SEQ ID NO: 129) CACNA1E voltage gated calciumchannel NM_000721 (Hs), NM_009782 6814 NP_000712 (Hs) (SEQ ID NO: 130),R-type a1E subunit (Mm), NM_019294 (Rn) NP_033912 (Mm) (SEQ ID NO: 131),NP_062167 (Rn) (SEQ ID NO: 132) CACNA1G voltage gated calcium channelNM_198378 (Hs), NM_198387 7135 NP_061496 (Hs) (SEQ ID NO: 133), T-typea1G subunit (Hs), NM_198379 (Hs), NP_938192 (Hs) (SEQ ID NO: 134),NM_938190 (Hs), NM_009783 NP_938190 (Hs) (SEQ ID NO: 135), (Mm),NM_031601 (Rn) NP_938201 (Hs) (SEQ ID NO: 136), NP_938193 (Hs) (SEQ IDNO: 137), NP_033913 (Mm) (SEQ ID NO: 138), NP_113789 (Rn) (SEQ ID NO:139) CACNA1H voltage gated calcium channel NM_021098 (Hs), NM_0214157069 NP_066921 (Hs) (SEQ ID NO: 140), T-type a1H subunit (Mm), NM_153814(Rn) NP_067390 (Mm) (SEQ ID NO: 141), NP_722521 (Rn) (SEQ ID NO: 142)CACNA1I voltage gated calcium channel NM_001003406 (Hs), 6673 NP_066919(Hs) (SEQ ID NO: 143), T-type a1I subunit NM_021096 (Hs), NP_001003406(Hs) (SEQ ID NO: NM_001044308 (Mm), 144), NP_001037773 (Mm) (SEQ IDNM_020084 (Rn) NO: 145), NP_064469 (Rn) (SEQ ID NO: 146) Voltage gatedpotassium channels Shaker voltage gated Potassium channel NM_000217(Hs), NM_010595 1492 NP_000208 (Hs) (SEQ ID NO: 147), KCNA1 DelayedRectifier member 1 (Mm), NM_173095 (Rn) NP_034725 (Mm), NP_775118 (Rn)Shaker voltage gated Potassium channel NM_004974 (Hs), NM_008417 1504NP_004965 (Hs) (SEQ ID NO: 148), KCNA2 Delayed Rectifier member 2 (Mm),NM_012970 (Rn) NP_032443 (Mm), NP_037102 (Rn) Shaker voltage gatedPotassium channel NM_002232 (Hs), NM_008418 1729 NP_002223 (Hs) (SEQ IDNO: 149), KCNA3 Delayed Rectifier member 3 (Mm), NM_019270 (Rn)NP_032444 (Mm), NP_062143 (Rn) Shaker voltage gated Potassium channelNM_002233 (Hs), NM_021275 1963 NP_002224 (Hs) (SEQ ID NO: 150), KCNA4A-Type, Fast inactivation (Mm), NM_012971 (Rn) NP_067250 (Mm), NP_037103(Rn) member 4 Shaker voltage gated Potassium channel NM_002234 (Hs),NM_145983 1843 NP_002225 (Hs) (SEQ ID NO: 151), KCNA5 Delayed Rectifiermember 5 (Mm), NM_012972 (Rn) NP_666095 (Mm), NP_037104 (Rn) Shakervoltage gated Potassium channel NM_002235 (Hs), NM_013568 1597 NP_002226(Hs) (SEQ ID NO: 152), KCNA6 Delayed Rectifier member 6 (Mm), NM_023954(Rn) NP_038596 (Mm), NP_076444 (Rn) Shaker voltage gated Potassiumchannel NM_031886 (Hs), NM_010596 1375 NP_114092 (Hs) (SEQ ID NO: 153),KCNA7 Delayed Rectifier member 7 (Mm), NM_001108914 (Rn) NP_034726 (Mm),NP_001102384 (Rn) Shaker voltage gated Potassium channel NM_005549 (Hs),1537 NP_005540 (Hs) (SEQ ID NO: 154), KCNA10 Delayed Rectifier member 10NM_001074609 (Mm), NP_001074609 (Mm), XP_227577 XM_227577 (Rn) (Rn) Shabvoltage gated Potassium channel NM_004975 (Hs), NM_008420 2581 NP_004966(Hs) (SEQ ID NO: 155), KCNB1 Delayed Rectifier Shab Related (Mm),NM_013186 (Rn) NP_032446 (Mm), NP_037318 (Rn) member 1 Shab voltagegated Potassium channel NM_004770 (Hs), 2737 NP_004761 (Hs) (SEQ ID NO:156), KCNB2 Delayed Rectifier Shab Related NM_001098528 (Mm),NP_001091998 (Mm), NP_446452 member 2 NM_054000 (Rn) (Rn) Shaw voltagegated Potassium channel NM_004976 (Hs), NM_008421 1537 NP_004967 (Hs)(SEQ ID NO: 157), KCNC1 Delayed Rectifier Shaw Related (Mm),NM_001112739 (Mm), NP_032447 (Mm), NP_001106210 member 1 NM_139217.1(Rn), (Mm), NP_631963 (Rn), NP_036988 NM_012856 (Rn) (Rn) Shaw voltagegated Potassium channel NM_153748 (Hs), NM_139136 1843 NP_631875 (Hs)(SEQ ID NO: 158), KCNC2 Delayed Rectifier Shaw Related (Hs), NM_139137(Hs), NP_715624 (Hs) (SEQ ID NO: 159), member 2 NM_001025581 (Mm),NP_631874 (Hs) (SEQ ID NO: 160), NM_139217 (Rn), NM_139216 NP_001020752(Mm), NP_631962 (Rn) (Rn), NP_631963 (Rn) Shaw voltage gated Potassiumchannel NM_004977 (Hs), NM_008422 2278 NP_004968 (Hs) (SEQ ID NO: 161),KCNC3 A-Type Shaw Related member 3 (Mm), NM_053997 (Rn) NP_032448 (Mm),NP_446449 (Rn) Shaw voltage gated Potassium channel NM_004978 (Hs),NM_145922 1912 NP_004969 (Hs) (SEQ ID NO: 162), KCNC4 A-Type Fastinactivation Shaw (Mm) NP_666034 (Mm), NP_001116248 Related member 4(Rn) Shal voltage gated Potassium channel NM_004979 (Hs), NM_008423 1948NP_004970 (Hs) (SEQ ID NO: 163), KCND1 A-Type Shal Related member 1(Mm), NM_001105748 (Rn) NP_032449 (Mm), NP_001099218 (Rn) Shal voltagegated Potassium channel NM_012281 (Hs), NM_019697 1894 NP_036413 (Hs)(SEQ ID NO: 164), KCND2 A-Type Shal Related member 2 (Mm), NM_031730(Rn) NP_062671 (Mm), NP_113918 (Rn) Shal voltage gated Potassium channelNM_172198 (Hs), NM_004980 1969 NP_004971 (Hs) (SEQ ID NO: 165), KCND3A-Type Shal Related member 3 (Hs), NM_019931 (Mm), NP_751948 (Hs) (SEQID NO: 166), NM_031739 (Rn) NP_064315 (Mm), NP_113927 (Rn) Modifiervoltage gated Potassium channel NM_002236 (Hs), NM_201531 1486 NP_002227(Hs) (SEQ ID NO: 167), KCNF1 modifier of Kv2 (Mm), NM_001169104 (Rn)NP_963289 (Mm), NP_001162575 (Rn) Modifier voltage gated Potassiumchannel NM_002237 (Hs), 1546 NP_002228 (Hs) (SEQ ID NO: 168), KCNG1modifier/silencer of Kv2 NM_001081134 (Mm), NP_001074603 (Mm),NP_001100015 NM_001106545 (Rn) (Rn) Modifier voltage gated Potassiumchannel NM_012283 (Hs), 1403 NP_036415.1 (Hs) (SEQ ID NO: 169), KCNG2modifier/silencer of Kv2 NM_012283.1 (Hs), NP_036415 (Hs) (SEQ ID NO:170), XM_140499 (Mm), XP_140499 (Mm), NP_001100842 NM_001107372 (Rn)(Rn) Modifier voltage gated Potassium channel NM_133329 (Hs), NM_1723441315 NP_579875 (Hs) (SEQ ID NO: 171), KCNG3 modifier/silencer of Kv2(Hs), NM_153512 (Mm), NP_758847 (Hs) (SEQ ID NO: 172), NM_001033957(Rn), NP_705732 (Mm), NP_596917 (Rn), NM_133426 (Rn) NP_001029129 (Rn)Modifier voltage gated Potassium channel NM_172347 (Hs), NM_025734 1564NP_758857 (Hs) (SEQ ID NO: 173), KCNG4 modifier/silencer of Kv2 (Mm),NM_001107435 (Rn) NP_080010 (Mm), NP_001100905 (Rn) KCNQ1 voltage gatedPotassium channel NM_000218 (Hs), NM_181798 2032 NP_000209 (Hs) (SEQ IDNO: 174), Delayed Rectifier KQT-Like (Hs), NM_008434 (Mm), NP_032460(Mm), NP_114462 (Rn) member 1 NM_032073 (Rn) KCNQ2 voltage gatedPotassium channel NM_172107 (Hs), NM_010611 2620 NP_742105 (Hs) (SEQ IDNO: 175), Delayed Rectifier KQT-Like (Mm), NM_133322 (Rn) NP_034741(Mm), NP_579856 (Rn) member 2 KCNQ3 voltage gated Potassium channelNM_004519 (Hs), NM_152923 2620 NP_004510 (Hs) (SEQ ID NO: 176), DelayedRectifier KQT-Like (Mm), NM_031597 (Rn) NP_690887 (Mm), NP_113785 (Rn)member 3 KCNQ4 voltage gated Potassium channel NM_172163.1 (Hs), 2089NP_751895 (Hs) (SEQ ID NO: 177), Delayed Rectifier KQT-Like NM_004700(Hs), NP_004691 (Hs) (SEQ ID NO: 178), member 4 NM_001081142 (Mm),NP_001074611 (Mm), XP_233477 XM_233477 (Rn) (Rn) KCNQ5 voltage gatedPotassium channel NM_019842 (Hs), NM_023872 2803 NP_062816 (Hs) (SEQ IDNO: 179), modifier/silencer of Kv2 (Mm), XM_001071249 (Rn) NP_076361(Mm), XP_001071249 (Rn) KCNV1 voltage gated Potassium channel NM_014379(Hs), NM_026200 1507 NP_055194 (Hs) (SEQ ID NO: 180), modifier/silencerof Kv2 (Mm), NM_021697 (Rn) NP_080476 (Mm), NP_067729 (Rn) KCNV2 voltagegated Potassium channel NM_133497 (Hs), NM_183179 1642 NP_598004 (Hs)(SEQ ID NO: 181), modifier/silencer of Kv2 (Mm), NM_001106370 (Rn)NP_899002 (Mm), NP_001099840 (Rn) KCNS1 voltage gated Potassium channelNM_002251 (Hs), NM_008435 1585 NP_002242 (Hs) (SEQ ID NO: 182),modifier/silencer of Kv2 (Mm), NM_053954 (Rn) NP_032461 (Mm), NP_446406(Rn) KCNS2 voltage gated Potassium channel NM_020697 (Hs), NM_1813171438 NP_065748 (Hs) (SEQ ID NO: 183), modifier/silencer of Kv2 (Mm),NM_023966 (Rn) NP_851834 (Mm), NP_076456 (Rn) KCNS3 voltage gatedPotassium channel NM_002252 (Hs), NM_173417 1477 NP_002243 (Hs) (SEQ IDNO: 184), modifier/silencer of Kv2 (Mm), NM_031778 (Rn) NP_775593 (Mm),NP_113966 (Rn) KCNH1 voltage gated Potassium channel NM_172362 (Hs),NM_010600 2974 NP_758872 (Hs) (SEQ ID NO: 185), Delayed RectifierEther-A-Gogo (Mm), NM_031742 (Rn) NP_034730 (Mm), NP_113930 (Rn) (eag1)KCNH5 voltage gated Potassium channel NM_172375 (Hs), NM_172376 2971NP_758963 (Hs) (SEQ ID NO: 186), Outward-Rectifying Ether-A- (Hs),NM_139318 (Hs), NP_758964 (Hs) (SEQ ID NO: 187), Gogo (eag2) NM_172805(Mm), NM_133610 NP_647479 (Hs), (Rn) NP_766393 (Mm), NP_598294 (Rn)KCNH2 voltage gated Potassium channel NM_172056 (Hs), NM_000238 3484NP_000229 (Hs) (SEQ ID NO: 188), Inwardly-Rectifying Ether-A- (Hs),NM_172057 (Hs), NP_742053 (Hs) (SEQ ID NO: 189), Gogo (erg1 or HERG)NM_013569 (Mm), NM_053949 NP_742054 (Hs) (SEQ ID NO: 190), (Rn)NP_038597 (Mm), NP_446401 (Rn) KCNH6 voltage gated Potassium channelNM_030779 (Hs), NM_173092 2989 NP_110406 (Hs) (SEQ ID NO: 191),Inwardly-Rectifying Ether-A- (Hs), NM_001037712 (Mm), NP_775115 (Hs)(SEQ ID NO: 192), Gogo (erg2) NM_053937 (Rn) NP_001032801 (Mm),NP_446389 (Rn) KCNH7 voltage gated Potassium channel NM_173162 (Hs),NM_033272 3595 NP_775185 (Hs) (SEQ ID NO: 193), Inwardly-RectifyingEther-A- (Hs), NM_133207 (Mm), NP_150375 (Hs) (SEQ ID NO: 194), Gogo(erg3) NM_131912 (Rn) NP_573470 (Mm), NP_571987 (Rn) KCNH8 voltage gatedPotassium channel NM_144633 (Hs), 3328 NP_653234 (Hs) (SEQ ID NO: 195),Inwardly-Rectifying Ether-A- NM_001031811 (Mm), NP_001026981 (Mm),NP_659563 Gogo (elk1) NM_145095 (Rn) (Rn) KCNH3 voltage gated Potassiumchannel NM_012284 (Hs), NM_010601 3259 NP_036416 (Hs) (SEQ ID NO: 196),Inwardly-Rectifying Ether-A- (Mm), NM_017108 (Rn) NP_034731 (Mm),NP_058804 (Rn) Gogo (elk2) KCNH4 voltage gated Potassium channelNM_012285 (Hs), 3058 NP_036417 (Hs) (SEQ ID NO: 197),Inwardly-Rectifying Ether-A- NM_001081194 (Mm), NP_001074663 (Mm),NP_446082 Gogo (elk1) NM_053630 (Rn) (Rn) Intracellularcalcium-activated potassium channels KCNMA1 large conductance calcium-NM_001014797 (Hs), 3550 NP_001014797 (Hs) (SEQ ID NO: activatedPotassium channel NM_010610 (Mm), NM_031828 198), NP_034740 (Mm),NP_114016 Slo1 (Rn) (Rn) KCNN1 small conductance calcium- NM_002248(Hs), NM_032397 1636 NP_002239 (Hs) (SEQ ID NO: 199), activatedPotassium channel (Mm), NM_019313 (Rn) NP_115773 (Mm), NP_062186 (Rn)SK1 KCNN2 small conductance calcium- NM_170775 (Hs), NM_021614 1744NP_740721 (Hs) (SEQ ID NO: 200), activated Potassium channel (Hs),NM_080465 (Mm), NP_067627 (Hs) (SEQ ID NO: 201), SK2 NM_019314 (Rn)NP_536713 (Mm), NP_062187 (Rn) KCNN3 small conductance calcium-NM_002249 (Hs), NM_170782 2197 NP_740752 (Hs) (SEQ ID NO: 202),activated Potassium channel (Hs), NM_080466 (Mm), NP_002240 (Hs) (SEQ IDNO: 203), SK3 NM_019315 (Rn) NP_536714 (Mm), NP_062188 (Rn) KCNN4intermediate conductance NM_002250 (Hs), NM_008433 1285 NP_002241 (Hs)(SEQ ID NO: 204), calcium-activated Potassium (Mm), NM_023021 (Rn)NP_032459 (Mm), NP_075410 (Rn) channel SK4 KCNT1 sodium activatedPotassium NM_020822 (Hs), NM_175462 3775 NP_065873 (Hs) (SEQ ID NO:205), channel Slo2.2 (Slack) (Mm), NM_021853 (Rn) NP_780671 (Mm),NP_068625 (Rn) KCNT2 sodium activated Potassium NM_198503 (Hs), 3409NP_940905 (Hs) (SEQ ID NO: 206), channel Slo2.1 (Slick) NM_001081027(Mm), NP_001074496 (Mm), NP_942057 NM_198762 (Rn) (Rn) KCNU1 sodiumactivated Potassium NM_001031836 (Hs), 3454 NP_001027006 (Hs) (SEQ IDNO: channel Slo3 NM_008432 (Mm) 207), NP_032458 (Mm) Inwardly rectifyingpotassium channels KCNJ1 Inwardly rectifying potassium NM_000220 (Hs),NM_153764 1180 NP_722448 (Hs) (SEQ ID NO: 208), channel Romk1 (Hs),NM_019659 (Mm), NP_000211 (Hs) (SEQ ID NO: 209), NM_017023 (Rn)NP_062633 (Mm), NP_058719 (Rn) KCNJ2 Inwardly rectifying potassiumNM_000891 (Hs), NM_008425 1285 NP_000882 (Hs) (SEQ ID NO: 210), channelIRK1 (Mm), NM_017296 (Rn) NP_032451 (Mm), NP_058992 (Rn) KCNJ12 Inwardlyrectifying potassium NM_021012 (Hs), NM_010603 1306 NP_066292 (Hs) (SEQID NO: 211), channel IRK2 (Mm), NM_053981 (Rn) NP_034733 (Mm), NP_446433(Rn) KCNJ4 Inwardly rectifying potassium NM_152868 (Hs), NM_008427 1342NP_690607 (Hs) (SEQ ID NO: 212), channel IRK3 (Mm), NM_053870 (Rn)NP_032453 (Mm), NP_446322 (Rn) KCNJ14 Inwardly rectifying potassiumNM_170720 (Hs), NM_145963 1312 NP_733838 (Hs) (SEQ ID NO: 213), channelIRK4 (Mm), NM_170718 (Rn) NP_666075 (Mm), NP_733836 (Rn) KCNJ3 Inwardlyrectifying potassium NM_002239 (Hs), NM_008426 1510 NP_002230 (Hs) (SEQID NO: 214), channel GIRK1 (Mm), NM_031610 (Rn) NP_032452 (Mm),NP_113798 (Rn) KCNJ6 Inwardly rectifying potassium NM_002240 (Hs),NM_010606 1273 NP_002231 (Hs) (SEQ ID NO: 215), channel GIRK2 (Mm),NM_001025590 (Mm), NP_001020756 (Mm), NP_001020755 NM_001025585 (Mm),(Mm), NP_034736 (Mm), NM_001025584 (Mm), NP_001020761 (Mm), NP_037324NM_013192 (Rn) (Rn) KCNJ9 Inwardly rectifying potassium NM_004983 (Hs),NM_008429 1186 NP_004974 (Hs) (SEQ ID NO: 216), channel GIRK3 (Mm),NM_053834 (Rn) NP_032455 (Mm), NP_446286 (Rn) KCNJ5 Inwardly rectifyingpotassium NM_000890 (Hs), NM_010605 1264 NP_000881 (Hs) (SEQ ID NO:217), channel GIRK4 (Mm), NM_017297 (Rn) NP_034735 (Mm), NP_058993 (Rn)KCNJ10 Inwardly rectifying potassium NM_002241 (Hs), 1144 NP_002232 (Hs)(SEQ ID NO: 218), channel BIR10 NM_001039484 (Mm), NP_001034573 (Mm),NP_113790 NM_031602 (Rn) (Rn) KCNJ15 Inwardly rectifying potassiumNM_170736 (Hs), 1129 NP_733932 (Hs) (SEQ ID NO: 219), channel IRKK(Kir1.3) NM_001039057 (Mm), NP_001034145 (Mm), NP_001034146 NM_001039056(Mm), (Mm), NP_579855 (Rn) NM_133321 (Rn) KCNJ16 Inwardly rectifyingpotassium NM_018658 (Hs), NM_010604 1261 NP_061128 (Hs) (SEQ ID NO:220), channel BIR9 (Mm), NM_053314 (Rn) NP_034734 (Mm), NP_445766 (Rn)KCNJ8 ATP-sensitive potassium NM_004982 (Hs), NM_008428 1279 NP_004973(Hs) (SEQ ID NO: 221), channel K-ATP1 (Mm), NM_017099 (Rn) NP_032454(Mm), NP_058795 (Rn) KCNJ11 ATP-sensitive potassium NM_000525 (Hs),NM_010602 1177 NP_000516 (Hs) (SEQ ID NO: 222), channel BIR (Mm),NM_031358 (Rn) NP_034732 (Mm), NP_112648 (Rn) KCNJ13 ATP-sensitivepotassium NM_002242 (Hs), NM_053608 1084 NP_002233 (Hs) (SEQ ID NO:223), channel (Kir1.4) (Rn) NP_446060 (Rn) 2-P-domain potassium channelsKCNK1 two pore domain potasium NM_002245 (Hs), NM_008430 1008 NP_002236(Hs) (SEQ ID NO: 224), channel TWIK1 (Mm), NM_021688 (Rn) NP_032456(Mm), NP_067720 (Rn) KCNK2 two pore domain potasium NM_001017424 (Hs),1278 NP_001017424 (Hs) (SEQ ID NO: channel TREK1 NM_010607 (Mm),NM_172042 225), NP_034737 (Mm), NP_742038 (Rn) (Rn) KCNK3 two poredomain potasium NM_002246 (Hs), NM_010608 1182 NP_002237 (Hs) (SEQ IDNO: 226), channel TASK1 (Mm), NM_033376 (Rn) NP_034738 (Mm), NP_203694(Rn) KCNK4 two pore domain potasium NM_033310 (Hs), NM_008431 1179NP_201567 (Hs) (SEQ ID NO: 227), channel TRAAK (Mm), NM_053804 (Rn)NP_032457 (Mm), NP_446256 (Rn) KCNK5 two pore domain potasium NM_003740(Hs), NM_021542 1497 NP_003731 (Hs) (SEQ ID NO: 228), channel TASK2(Mm), NM_001039516 (Rn) NP_067517 (Mm), NP_001034605 (Rn) KCNK6 two poredomain potasium NM_004823 (Hs), 939 NP_004814 (Hs) (SEQ ID NO: 229),channel TWIK2 NM_001033525 (Mm), NP_001028697 (Mm), NP_446258 NM_053806(Rn) (Rn) KCNK7 two pore domain potasium NM_033347 (Hs), NM_010609 921NP_203133 (Hs) (SEQ ID NO: 230), channel member 7 (Mm) NP_034739 (Mm)KCNK9 two pore domain potasium NM_016601 (Hs), 1122 NP_057685 (Hs) (SEQID NO: 231), channel TASK3 NM_001033876 (Mm), NP_001029048 (Mm),NP_445857 NM_053405 (Rn) (Rn) KCNK10 two pore domain potasium NM_021161(Hs), NM_029911 1614 NP_066984 (Hs) (SEQ ID NO: 232), channel TREK2(Mm), NM_023096 (Rn) NP_084187 (Mm), NP_075584 (Rn) KCNK12 two poredomain potasium NM_022055 (Hs), NM_199251 1290 NP_071338 (Hs) (SEQ IDNO: 233), channel THIK2 (Mm), NM_022292 (Rn) NP_954859 (Mm), NP_071628(Rn) KCNK13 two pore domain potasium NM_022054 (Hs), NM_146037 1224NP_071337 (Hs) (SEQ ID NO: 234), channel THIK1 (Mm), NM_022293 (Rn)NP_666149 (Mm), NP_071629 (Rn) KCNK15 two pore domain potasium NM_022358(Hs), 990 NP_071753 (Hs) (SEQ ID NO: 235), channel TASK5 NM_001030292(Mm), NP_001025463 (Mm), NP_570826 NM_130813 (Rn) (Rn) KCNK16 two poredomain potasium NM_032115 (Hs), NM_029006 930 NP_115491 (Hs) (SEQ ID NO:236), channel TALK1 (Mm) NP_083282 (Mm) KCNK17 two pore domain potasiumNM_031460 (Hs) 996 NP_113648 (Hs) (SEQ ID NO: 237) channel TASK4-TALK2KCNK18 two pore domain potasium NM_181840 (Hs), NM_207261 1152 NP_862823(Hs) (SEQ ID NO: 238), channel TRESK1/TRESK2 (Mm), NM_001003820 (Rn)NP_997144 (Mm), NP_001003820 (Rn) Nucleotide-activated ion channelsCNGA1 Cyclic nucleotide gated cation NM_000087 (Hs), NM_007723 2073NP_000078 (Hs) (SEQ ID NO: 239), channel Alpha 1 (Mm), NM_053497 (Rn)NP_031749 (Mm), NP_445949 (Rn) CNGA2 Cyclic nucleotide gated cationNM_005140 (Hs), NM_007724 1995 NP_005131 (Hs) (SEQ ID NO: 240), channelAlpha 2 (Mm), NM_012928 (Rn) NP_031750 (Mm), NP_037060 (Rn) CNGA3 Cyclicnucleotide gated cation NM_001298 (Hs), NM_009918 2086 NP_001073347 (Hs)(SEQ ID NO: channel Alpha 3 (Mm), NM_053495 (Rn) 241), NP_034048 (Mm),NP_445947 (Rn) CNGA4 Cyclic nucleotide gated cation NM_001037329 (Hs),1731 NP_001032406 (Hs) (SEQ ID NO: channel Alpha 4 NM_001033317 (Mm),242), NP_001028489 (Mm), NM_053496 (Rn) NP_445948 (Rn) CNGB1 Cyclicnucleotide gated cation NM_001297 (Hs), NM_031809 3763 NP_001288 (Hs)(SEQ ID NO: 243), channel Beta1 (Rn) XP_001476248 (Mm), NP_113997 (Rn)CNGB3 Cyclic nucleotide gated cation NM_019098 (Hs), NM_013927 2434NP_061971 (Hs) (SEQ ID NO: 244), channel Beta3 (Mm) NP_038955 (Mm) HCN1hyperpolarization Cyclic NM_021072 (Hs), NM_010408 2674 NP_066550 (Hs)(SEQ ID NO: 245), nucleotide gated cation channel (Mm), NM_053375 (Rn)NP_034538 (Mm), NP_445827 (Rn) member 1 HCN2 hyperpolarization CyclicNM_001194 (Hs), NM_008226 2671 NP_001185 (Hs) (SEQ ID NO: 246),nucleotide gated cation channel (Mm), NM_053684 (Rn) NP_032252 (Mm),NP_446136 (Rn) member 2 HCN3 hyperpolarization Cyclic NM_020897 (Hs),NM_008227 2326 NP_065948 (Hs) (SEQ ID NO: 247), nucleotide gated cationchannel (Mm), NM_053685 (Rn) NP_032253 (Mm), NP_446137 (Rn) member 3HCN4 hyperpolarization Cyclic NM_005477 (Hs), 3613 NP_005468 (Hs) (SEQID NO: 248), nucleotide gated cation channel NM_001081192 (Mm),NP_001074661 (Mm), NP_067690 member 4 NM_021658 (Rn) (Rn) TRP channelsTRPC1 transient receptor potential NM_003304 (Hs), NM_011643 2284NP_003295 (Hs) (SEQ ID NO: 249), (TRP) non selective cation (Mm),NM_053558 (Rn) NP_035773 (Mm), NP_446010 (Rn) channel C member 1 TRPC3diacyglycerol (DAG) Ca2+ NM_003305 (Hs), NM_019510 2548 NP_003296 (Hs)(SEQ ID NO: 250), activated TRP channel C (Mm), NM_021771 (Rn) NP_062383(Mm), NP_068539 (Rn) TRPC4 TRP receptor operated channel NM_016179 (Hs),NM_016984 2935 NP_057263 (Hs) (SEQ ID NO: 251), (Mm), NM_080396 (Rn),NP_058680 (Mm), NP_001076584 NM_001083115 (Rn) (Rn), NP_536321 (Rn)TRPC5 TRP receptor operated channel NM_012471 (Hs), NM_009428 2923NP_036603 (Hs) (SEQ ID NO: 252), (Mm), NM_080898 (Rn) NP_033454 (Mm),NP_543174 (Rn) TRPC6 DAG activated TRP channel NM_004621 (Hs), NM_0138382800 NP_004612 (Hs) (SEQ ID NO: 253), (Mm), NM_053559 (Rn) NP_038866(Mm), NP_446011 (Rn) TRPC7 Cardiac TRP channel NM_020389 (Hs), NM_0120352593 NP_065122 (Hs) (SEQ ID NO: 254), (Mm), XM_225159 (Rn) NP_036165(Mm), XP_001067646 (Rn) TRPV1 Vanilloid (capsaicin) receptor NM_018727(Hs), 2524 NP_061197 (Hs) (SEQ ID NO: 255), and noxious thermosensorNM_001001445 (Mm), NP_001001445 (Mm), NP_114188 channel NM_031982 (Rn)(Rn) TRPV2 noxious heat thermosensor NM_016113 (Hs), NM_011706 2299NP_057197 (Hs) (SEQ ID NO: 256), channel (Mm), NM_017207 (Rn) NP_035836(Mm), NP_58903 (Rn) TRPV3 warmth sensor channel NM_145068 (Hs),NM_145099 2380 NP_659505 (Hs) (SEQ ID NO: 257), (Mm), NM_001025757 (Rn)NP_659567 (Mm), NP_001020928 (Rn) TRPV4 Osmosensor channel NM_021625(Hs), NM_022017 2620 NP_067638 (Hs) (SEQ ID NO: 258), (Mm), NM_023970(Rn) NP_071300 (Mm), NP_76460 (Rn) TRPM1 putative melastatin TRP channelNM_002420 (Hs), NM_018752 4816 NP_002411 (Hs) (SEQ ID NO: 259), (Mm),NM_001037734 (Rn) NP_061222 (Mm), NP_001032823 (Rn) TRPM2 nucleotidesensing TRP channel NM_003307 (Hs), NM_138301 4513 NP_003298 (Hs) (SEQID NO: 260), (Mm), NM_001011559 (Rn) NP_612174 (Mm), NP_001011559 (Rn)TRPM3 Melastatin related TRP channel NM_020952 (Hs), 4669 NP_066003 (Hs)(SEQ ID NO: 261), NM_001035239 (Mm), NP_001030319 (Mm), XP_219902XM_219902 (Rn) (Rn) TRPM4 Ca2+ activated Na+ TRP NM_017636 (Hs),NM_175130 3646 NP_060106 (Hs) (SEQ ID NO: 262), channel (Mm),NM_001136229 (Rn) NP_780339 (Mm), NP_001129701 (Rn) TRPM5 Nonselective,monovalent cation NM_014555 (Hs), NM_020277 3502 NP_055370 (Hs) (SEQ IDNO: 263), TRP channel (Mm), XM_344979 (Rn) NP_064673 (Mm), XP_344980(Rn) TRPM6 Channel kinase TRP channel NM_017662 (Hs), NM_153417 6073NP_060132 (Hs) (SEQ ID NO: 264), (Mm), XM_219747 (Rn) NP_700466 (Mm),XP_219747 (Rn) TRPM7 Kinase TRP channel NM_017672 (Hs), NM_021450 5602NP_060142 (Hs) (SEQ ID NO: 265), (Mm), XM_001056331 (Rn) NP_067425 (Mm),XP_001056331 (Rn) TRPM8 Cooling and menthol-sensing NM_024080 (Hs),NM_134252 3319 NP_076985 (Hs) (SEQ ID NO: 266), TRP channel (Mm),NM_134371 (Rn) NP_599013 (Mm), NP_599198 (Rn) TRPA1 Nonselective cationTRP NM_007332 (Hs), NM_177781 3364 NP_015628 (Hs) (SEQ ID NO: 267),channel (Mm), NM_207608 (Rn) NP_808449 (Mm), NP_997491 (Rn) TRPP1Nonselective cation TRP NM_000297 (Hs), NM_008861 2908 NP_000288 (Hs)(SEQ ID NO: 268), channel PKD2 (Mm), XM_573552 (Rn) NP_032887 (Mm),XP_573552 (Rn) TRPP2 Nonselective cation TRP NM_016112 (Hs), NM_1814222422 NP_057196 (Hs) (SEQ ID NO: 269), channel PKD2L1 (Mm), NM_001106352(Rn) NP_852087 (Mm), NP_001099822 (Rn) TRPP3 Nonselective cation TRPNM_014386 (Hs), NM_016927 1843 NP_055201 (Hs) (SEQ ID NO: 270), channelPKD2L2 (Mm), NM_001106156 (Rn) NP_058623 (Mm), NP_001099626 (Rn) TRPML1Nonselective cation TRP NM_020533 (Hs), NM_053177 1747 NP_065394 (Hs)(SEQ ID NO: 271), channel (Mm), NM_001105903 (Rn) NP_444407 (Mm),NP_001099373 (Rn) TRPML2 Nonselective cation TRP NM_153259 (Hs), 1702NP_694991 (Hs) (SEQ ID NO: 272), channel NM_001005846 (Mm), NP_001005846(Mm), NP_080932 NM_026656 (Mm), (Mm), NP_001034094 (Rn) NM_001039005(Rn) TRPML3 Nonselective cation TRP NM_018298 (Hs), NM_134160 1663NP_060768 (Hs) (SEQ ID NO: 273), channel (Mm), NM_001012059 (Rn)NP_598921 (Mm), NP_001012059 (Rn) Chloride channels CLC1 chloridechannel 1 NM_000083.2 2968 NP_000074.2 (SEQ ID NO: 274) CLC2 chloridechannel 2 NM_004366.4 2698 NP_004357.3 (SEQ ID NO: 275) CLC3 chloridechannel 3 NM_173872.3 2602 NP_776297.2 (SEQ ID NO: 276) CLC4 chloridechannel 4 NP_001821.2 2284 NP_001821.2 (SEQ ID NO: 277) CLC5 chloridechannel 5 NM_000084.2 2242 NP_000075.1 (SEQ ID NO: 278) CLC6 chloridechannel 6 NM_001286.2 2611 NP_001277.1 (SEQ ID NO: 279) CLC7 chloridechannel 7 NM_001287.4 2419 NP_001278.1 (SEQ ID NO: 280) Acid-sensitivecation channels ACCN1 amiloride-sensitive cation NM_183377.1 1696NP_899233.1 (SEQ ID NO: 281) channel 1 ACCN2 amiloride-sensitive cationNM_020039.2 1726 NP_064423.2 (SEQ ID NO: 282) channel 2 ACCN3amiloride-sensitive cation NM_020322.2 1600 NP_064718.1 (SEQ ID NO: 283)channel 3 ACCN4 amiloride-sensitive cation NM_018674.4 2005 NP_061144.3(SEQ ID NO: 284) channel 4 Sodium channel auxiliary subunits SCN1Bvoltage gated sodium channel NM_001037.4 658 NP_001028.1 (SEQ ID NO:285) type I b subunit SCN2B voltage gated sodium channel NM_004588.4 652NP_004579.1 (SEQ ID NO: 286) type 2 b subunit SCN3B voltage gated sodiumchannel NM_018400.3 652 NP_060870.1 (SEQ ID NO: 287) type 3 b subunitSCN4B voltage gated sodium channel NM_174934.3 694 NP_777594.1 (SEQ IDNO: 288) type 4 b subunit Calcium channel auxiliary subunits CACNB1voltage gated calcium channel NM_000723.4 1798 NP_000714.3 (SEQ ID NO:289) b1 subunit CACNB2 voltage gated calcium channel NM_000724.3 1822NP_000715.2 (SEQ ID NO: 290) b2 subunit CACNB3 voltage gated calciumchannel NM_000725.3 1459 NP_000716.2 (SEQ ID NO: 291) b3 subunit CACNB4voltage gated calcium channel NM_000726.3 1465 NP_000717.2 (SEQ ID NO:292) b4 subunit CACNA2 voltage gated calcium channel NM_000722.2 (HS),3277 NP_000713.2 (HS) (SEQ ID NO: 293), a2d subunit NM_001110843.1 (Mm)NP_001104313.1 (Mm), NP_037051 NM_012919 (RN) (RN) CACNG2 voltage gatedcalcium channel g NM_006078.3 (HS), 973 NP_006069.1 (HS) (SEQ ID NO:294), subunit NM_007583.2 (Mm) NP_031609.1 (Mm) OTHERS ABCC8 ATP-bindingcassette, sub- NM_000352.3 (HS) NP_000343.2 (HS) (SEQ ID NO: 295),family C SUR1 NM_011510.3 (Mm) NP_035640.2 (Mm) ABCC9 SUR2 NM_020297.2NP_064693.2 (SEQ ID NO: 296) LGi1 LGi1 NM_005097.2 NP_005088.1 (SEQ IDNO: 297) CNTNAP2 CASPR2 NM_014141.5 NP_054860.1 (SEQ ID NO: 298) KCNAB1Kv b1 NM_172160.2 NP_751892.1 (SEQ ID NO: 299) KCNAB2 Kv b2 NM_003636.3NP_003627.1 (SEQ ID NO: 300) KCNAB3 Kv b3 NM_004732.2 NP_004723.2 (SEQID NO: 301) KCNMB1 BK b1 NM_004137.2 NP_004128.1 (SEQ ID NO: 302) KCNMB2BK b2 NM_005832.3 NP_005823.1 (SEQ ID NO: 303) KCNMB3 BK b3 NM_171828.1NP_741979.1 (SEQ ID NO: 304) KCNMB4 BK b4 NM_014505.5 NP_055320.4 (SEQID NO: 305) AQP1 AQP1 NM_198098.2 NP_932766.1 (SEQ ID NO: 306) AQP2 AQP2NM_000486.5 NP_000477.1 (SEQ ID NO: 307) AQP3 AQP3 NM_004925.4NP_004916.1 (SEQ ID NO: 308) AQP4 AQP4 NM_001650.4 NP_001641.1 (SEQ IDNO: 309) GJB1 Connexin 32 NM_000166.5, NM_000165.3 NP_000157.1 (SEQ IDNO: 310) GJA1 Connexin 43 NM_000165.3 NP_000156.1 (SEQ ID NO: 311) Hs:Homo sapiens (human) Mm: Mus musculus (mouse) Rn: Rattus norvegicus(rat)

In one particular embodiment of the invention, the membrane protein ofinterest is chosen from a GABA receptor, typically a GABA_(B)(GABA_(B)R1 or GABA_(B)R2 for example) receptor; a glutamate receptor,typically an alpha-amino-3-hydroxy-5-methylisoazole-4-propionic acid(AMPA) receptor, preferably the GluR3 subunit of the glutamate receptor(SEQ ID NO:73 or SEQ ID NO:74) or an N-methyl-D-aspartate(NMDA)-activated glutamate receptor, preferably the NMDAR1 subunit ofthe human glutamate receptor (SEQ ID NO:62); aquaporin 2 (SEQ IDNO:307); aquaporin 4 (SEQ ID NO:309); a potassium channel; a calciumchannel; an acetylcholine receptor, preferably the α3 subunit (SEQ IDNO:9) of the human nicotinic acetylcholine receptor (ACHα3); a proteinassociated with voltage gated potassium channels, preferably CASPR2 (SEQID NO:298) or LGI1 (SEQ ID NO:297); a glycine receptor; connexin 32 (SEQID NO:310) and connexin 43 (SEQ ID NO:311).

Another subject of the invention relates to a chip comprising at least20 different proteins of interest, for example at least 50, typically atleast 100 different proteins of interest, preferably betweenapproximately 20 and approximately 400 different proteins of interest,for example approximately 250 or approximately 300 different proteins ofinterest, or between approximately 20 and approximately 100 differentproteins of interest, for example approximately 50 different proteins ofinterest, each protein of interest being optionally associated with oneor more auxiliary subunits. These proteins of interest are preferablymembrane proteins chosen from the proteins of which the sequences(preferably the sequences of the human proteins) are identified in Table2.

The inventors have in particular developed chips comprising membraneproteins corresponding to proteins such as ion channel proteins,transporter proteins and/or membrane receptor proteins which regulatethe activity of an ion channel protein or of a transporter protein.These proteins are encoded by the sequences, preferably the sequencesidentified by a SEQ ID number, even more preferentially by the humansequences identified by a SEQ ID number, referenced in Table 2.

In order to fabricate these chips, the inventors have, without a priori,amplified by PCR a large number of subunits obtained from human braincDNA (see experimental section). The products have been subcloned intoan expression vector and transfected into cells in culture in multiwellplates, each well containing cells expressing a single type of membraneprotein or membrane protein complex. The same tag sequence introducedinto each channel makes it possible, in accordance with the knowledge ofthose skilled in the art, to measure and standardize the expressionlevel of the proteins or protein complexes. The proteins and proteincomplexes of these cells are solubilized in a buffer containing anon-denaturing detergent. This solubilisate is then typically depositedin the form of a spot on a support, preferably a solid support, forexample on a solid membrane. Typically, various amounts of solubilisate,and also preferably one or more negative and positive controls, aredeposited on the same support in order to verify the specificity of thesignals obtained. The reading of the chip (detection of theantigen/antibody (Ag/Ab) complexes) can be carried out byimmunofluorescence, immunoluminescence, or colorimetry by means of oneof the techniques known to those skilled in the art.

Thus, a particular subject of the invention relates to a chip asdescribed in the present text, said protein(s) of interest of which is(are) labelled with the same peptide sequence (tag), recognized by oneor more detection antibodies known to those skilled in the art, such as,for example, the V5 antibody which recognizes the GKPIPNPLLGLDSTsequence (SEQ ID NO:312). Other tags that can be used in the context ofthe present invention are, for example, the T7 Flag, Ha, myc and His(hexahistidine) tags.

In one preferred embodiment, the protein chip according to the inventioncomprises at least one positive control and/or one negative control,typically one positive control and one negative control, in order toverify the specificity of the signals obtained. The negative controltypically consists of a cell extract not comprising the protein ofinterest. The positive control typically consists of humanimmunoglobulins, for example IgGs, and makes it possible to validate theability of the protein chip according to the invention to detect thepresence of autoantibodies capable of binding to the proteins ofinterest in a biological sample to be tested.

The chips used in the context of the invention comprise a supportcapable of binding solubilized proteins, typically a solid (orsemi-solid) support, preferably chosen from a membrane, typically anitrocellulose or nylon membrane; glass; and a polymer capable ofabsorbing immune complexes, typically plastic (for example PVDF).

Such a support can be treated, in order to improve its cellextract-binding properties, using products and according to methodsknown to those skilled in the art. The support can, for example, becoated with a layer of organosilicon compounds (silane) and/or befunctionalized with glycosaminoglycans (GAGs).

Another subject of the invention relates, moreover, to a kit comprisinga chip as described previously, and, optionally, one or more reagentspreferably chosen from a buffer for blocking the non-specific sites, abuffer allowing the association between antibodies and antigens (Ab/Ag),a washing buffer, one or more secondary antibodies, one or moreproduct(s) for detecting said secondary antibody or antibodies, astandard solution for preparing a calibration curve, and instructionsfor use, typically instructions for use of one, several or all of saidelements of the kit.

Such a kit may typically be used in the screening for new targets ofinterest, for example targets involved in the occurrence of anautoimmune disease, in particular of a disease affecting the nervoussystem of a mammal, or in the diagnosis or the monitoring of theprogression of such an autoimmune disease in a patient receivingtreatment in order to determine, in this patient, the efficacy of saidtreatment.

One particular subject of the invention thus relates to a kit forscreening for targets of interest involved in the occurrence of adisease of interest, for example of an autoimmune disease.

Another particular subject of the invention relates, moreover, to a kitfor diagnosing a disease, typically an autoimmune disease as describedpreviously, in an animal, typically a mammal, preferably a human being.

Preparation of the Chip

Another subject of the invention relates to a method for preparing achip as described previously, comprising the following steps a) ofcloning the cDNA(s) of interest; b) of expressing the protein(s),typically the membrane protein(s), encoded by said cDNA(s) in aprotein-producing system; typically in cells in culture; c) ofsolubilizing the proteins expressed using a non-denaturing detergent,typically a non-ionic detergent (also identified as “mild detergent”),which enables the solubilization of said proteins while at the same timepreserving their conformation (three-dimensional structure) and theirability to bind to a support; and d) of depositing said solubilizedproteins on a support in order to obtain the chip of interest.

The invention also relates to a a chip capable of being obtained bymeans of a method according to the invention as described previously.

Preferably, the chip according to the invention allows the detection, bythose skilled in the art, of the proteins of interest which have boundto the support, by means of a conventional detection method, typicallyinvolving a single detection antibody. Such a chip allows its fabricatoror its user to be sure that the protein(s) of interest is correctlyexpressed on said chip.

A means for obtaining such a chip consists in cloning the protein ofinterest (step a) of the method described previously) using anexpression vector which allows i) the insertion of a tag, as describedpreviously, in the same reading frame as the sequence encoding theprotein of interest, and ii) the expression of a protein fused,preferably in the C-terminal position, to said tag. Such a tag makes itpossible to verify and to measure the expression of the protein ofinterest associated with said tag.

The expression vector used is preferably a directional vector whichallows the insertion of the gene of interest following PCR amplificationthereof, without a prior enzymatic digestion step. An example of adirectional vector is provided by the experimental section of thepresent application which uses the pcDNA6.2/V5/GW/D-TOPO vector(Invitrogen).

Advantageously, the vector used also benefits from the Gatewaytechnology which makes it possible to envisage the rapid transfer of thegene encoding the protein of interest, from a first vector to a secondvector benefiting from the same technology but having differentexpression properties (the gene being cloned into the donor vectorbetween two recombination sites compatible with all the Gatewayrecipient vectors). This technology makes it possible, for example, totransfer the gene encoding the protein of interest from a vector whichallows expression in mammalian cells (donor vector) to a vector(recipient vector) which allows expression in insect cells, batrachiancells, yeast or bacteria, or else to a vector that is identical but hasno tag. A vector benefiting from the Gateway technology can be chosen,for example, from the vectors pT-REx™-DEST30 (allowing expression inmammalian cells), pET™-DEST42 (allowing expression in bacteria), pDEST™8(allowing expression in insect cells) and pYES2-DEST52 (allowingexpression in yeasts).

During the PCR amplification of the genes, the forward primer preferablybegins with the sequence CACC (SEQ ID NO:313) (which precedes the startcodon (ATG) of the gene of interest). In another preferred embodiment, aguanidine (G) nucleotide is inserted after the ATG start codon in orderto obtain a Kozak sequence. In yet another embodiment, an additional GGAcodon is introduced into the nucleotide sequence following the startcodon of the gene of interest.

According to yet another preferred embodiment, the reverse primer doesnot contain a stop codon and/or does not end with the sequencecomplementary to the beginning of the forward primer.

Preferentially, the (forward and/or reverse) primers have ahybridization temperature of between approximately 70° C. andapproximately 80° C., and also advantageously a percentage of GC unitsof less than approximately 60%.

Step b) of expressing the protein(s) encoded by said cDNA(s) of interestis advantageously carried out in cells in culture, preferably in humancells. The cells are generally placed in culture on multiwell plates,optionally treated to promote the adhesion of the cells using thetechniques known to those skilled in the art, each well preferablycontaining cells expressing a single type of protein of interest. Theexpression of the protein of interest can be controlled as early as thisstage, and if required standardized, through the possible detection (forexample by immunofluorescence) of the tag sequence that is expressed,where appropriate at the same time as the protein of interest. The cellsselected are preferably cells exhibiting the satisfactory properties ofadhesion to the culture support, which are simple to culture and tomanipulate. Such cells can be advantageously selected, for example, fromthe HEK293A, COS, MDCK, CHO and CCL39 lines.

The method according to the invention comprises, moreover, a step c) ofsolubilizing the protein(s) of interest using a detergent, preferablyusing a non-denaturing detergent capable of preserving the conformationof the native protein and also its ability to bind to a support,typically to a support as described previously, preferably to a solidsupport of membrane type.

The inventors have discovered that the detergent can be advantageouslyselected from triton X_(—)100, triton X_(—)114, Nonidet P-40, CHAPS,sodium cholate, sodium deoxycholate, digitonin, sarkosyl NL30 andoctylglucoside.

Finally, the method comprises a step d) of depositing solubilizedproteins of interest onto a support as described previously, in order toobtain the chip according to the invention. This step is typicallycarried out by depositing, in the form of spots, the solubilisatesobtained at the end of step c) onto the chosen support, by means oftechniques known to those skilled in the art.

Uses

There is in particular at the current time no tool for simply andeffectively detecting the presence of autoantibodies directed againstmembrane proteins and/or membrane protein complexes, in a biologicalsample, without having to visualize by microscopy the antigens/antibody(Ag/Ab) complexes formed at the surface of cells (previously transfectedwith the sequence encoding the antigen of interest) or of tissuesections. The chip according to the invention is thus the firstavailable tool which allows the detection of Ag protein complexes ormembrane protein/Ab complexes directly on a solid support (test onmembrane for example).

The chip according to the invention can be advantageously used in thecontext of a method for the diagnosis and/or for the monitoring of theprogression of a disease, typically of an autoimmune disease asdescribed previously, preferably of an encephalopathy associated withneuronal excitability disorders, linked to the expression of one or moreof the proteins, in particular membrane proteins, of interest bound tothe support of said chip, or else in the context of a method fordetermining the efficacy of a treatment applied to a given patient. Theimmediate or gradual disappearance of the autoantibodies sought in thebiological sample originating from a patient receiving therapeutictreatment, evaluated using the protein chip according to the invention,thus makes it possible to verify, over time, the efficacy of thetreatment applied to said patient. The absence of an effect of saidtreatment, readily detectable using said chip, makes it possible,moreover, to then correct or adapt, at an early stage, the treatmentapplied to a given patient.

The chip according to the invention may also be used in the context ofmethods required for searching for and identifying (screening methods)antibodies of interest, even present at very low concentrations (i.e.about one millimole), in particular autoantibodies, and also new antigentargets of diseases, for example of autoimmune diseases (antigens ofinterest involved in the occurrence of an immune disease).

The biological samples from subjects identified as suffering frompathological conditions suspected of belonging or clearly identified asbelonging to the autoimmune disease category can advantageously be usedfor this purpose.

It is thus now possible to produce and use a chip according to theinvention [typically a chip which has i) all of the proteins, inparticular of the proteins or membrane protein complexes, expressed by agiven tissue, ii) all of the proteins, in particular of the proteins ormembrane protein complexes, involved in a particular physiologicalmechanism, or iii) all of the proteins, in particular of the proteins ormembrane protein complexes, involved in a specific metabolic pathway] inorder to detect new autoantibodies or to identify new targets oftherapeutic interest.

The large-scale screening permitted by this new tool allows thesimultaneous detection of a very large number of Ag/Ab complexes. Italso allows archiving of results which is simpler than that permitted bythe currently known tests used on cells or tissue sections. Themembranes represent surface areas of approximately 4 cm by 4 cm with thethickness of a sheet of paper. They are detected on a “photo” ofidentical size which can be stored like any paper or digital photo. Inthe case of tissue sections or transfected cells, the slides arearchived in a freezer.

The facilitated identification of antibodies, for example ofautoantibodies involved in autoimmune pathological conditions of thenervous system, and of their targets, using a chip according to theinvention offering sufficient sensitivity, presently allows thedevelopment of diagnostic tests, in particular of serological tests,which are more efficient, simpler and less expensive.

The protein chip according to the invention, by virtue of the newantigen/antibody complexes that it makes it possible to identify,allows, moreover, the development of new therapeutic treatmentsinvolving the use of the antibodies identified, or of modulators of theformation of said antigen/antibody complexes, as medicaments, or in thecontext of the preparation of pharmaceutical compositions of interest.

The following figures and examples illustrate the invention bydescribing the obtaining of chips in accordance with the invention andby providing examples of uses of said chips, without limiting the scopethereof.

FIGURE LEGENDS

FIG. 1: Validation of the efficiency of the protein chip by virtue of atest carried out on the samples originating from 43 patients.

A. Plan of a plate representing quadruplicates of a nitrocellulosemembrane onto which are deposited, for each of the quadruplicates,decreasing concentrations of an extract of solubilized membranes ofcells having overexpressed the NR1 receptor, an extract of solubilizedmembranes of cells without vector, and a 1/200^(th) dilution of humanserum.

B. Photos of supports corresponding, respectively, to i) an expressioncontrol using the V5 tag alone, ii) an expression control using a sampleoriginating from a positive patient, iii) an example of a negativesample, and iv) an example of a positive sample.

Of the 43 samples tested, 15 samples out of 17 positive samples weredetected using the protein chip, i.e. a protein chip specificity at 88%.100% of the negative samples were, moreover, detected.

FIG. 2: Example of use of a protein chip according to the inventioncomprising 40 proteins of interest.

A. Plate 1: it contains two wells containing human IgGs and 22 wellscontaining the solubilized proteins of HEK cell membranes, each cellexpressing a different membrane protein chosen from the proteinsidentified in table 2 (referenced SEQ ID NO:1 to SEQ ID NO:311).

B. Plate 2: it contains two wells containing human IgGs and 22 wellscontaining the solubilized proteins of HEK cell membranes, each cellexpressing a different membrane protein chosen from the proteinsidentified in table 2 (referenced SEQ ID NO:1 to SEQ ID NO:311) and notexpressed by the cells used to prepare plate 1.

C. Control plate (the correct expression is verified using the V5 tagalone)

D. Example of non-specific response

E. Example of negative response

F. Example of positive response

EXAMPLES Example 1 Preparation of a Protein Chip According to theInvention and Developing of the Proteins Bound Materials and MethodCloning

The inventors used the pcDNA6.2-TOPO/GW/V5 expression vector (InvitrogenCat#K 2460-20). The majority of the primers were ordered fromEurogentech and the rest from Sigma. The PCR is carried out on aThermocycler machine (Biometra) with a commercial mixture of polymerases(HF enzyme mix from Fermentas cat#K0192). The amplification protocol isof the 2-step type with an extension and amplification temperature at68° C.

The template for the amplification of the genes of interest is mainlyderived from a cDNA library prepared from mRNA of healthy human brain(Invitrogen). The reverse transcription of this mRNA into cDNA iscarried out using a Superscript III (Invitrogen) according to themaker's protocol.

In the event of failed amplification of the gene of interest (eitherbecause of too little expression of this gene in the brain and thereforeof the difficulty in amplifying it from this cDNA library, or because ofthe nucleotide nature of this gene, or else for another undeterminedreason), another source of RNA (human cells of HEK type for example) isused or a plasmid construct is used when it is available.

The PCR-amplified genes are separated from the dNTPs and from thetemplate by (1%) agarose gel electrophoresis. The DNA bands of molecularweights corresponding to the genes of interest are cut out of the geland purified on a column (of the “gel extraction purification” type fromQuiagen, Promega or Macherey Nagel). The amplified genes thus purifiedare then inserted into the expression vector according to the maker'sguidelines (TOPO-cloning, Invitrogen).

The plasmid constructs thus created are screened by bacterialtransformation. Plated out on bacterial culture dishes, they areselected with an antibiotic of ampicillin type. The positive bacteria(of TOP10 type, Invitrogen) are placed in liquid culture the next daywith the same antibiotic selection. This bacterial culture amplificationmakes it possible to amplify the amount of DNA available. This amplifiedDNA is purified on a column (of miniperp type, Qiagen). The plasmidsthat may have incorporated the gene of interest are sequenced using asequencing platform. The sequencing primers are the T7 forward primerand a reverse primer specific for the pcDNA6.2-TOPO/GW/V5 vector. Onlythe beginning and the end of the sequence of the gene are verified bysequencing. This is because the subsequent detection of the presence ofthe tag (ta-V5g) in the cells in culture will by itself attest to theintegrity of the protein.

Cell Culture

The inventors use the HEK293A human cell strain (Invitrogen Cat# R70507)as protein production system.

For the cell transfection, the inventors used JetPEI (Ozyme cat#101-10)as transfecting agent.

The inventors transfected a first series of cells on glass coverslipsprecoated with poly-L-Lysine in order to facilitate their adhesion.

Two days after the transfection, the inventors verify the presence ofthe Tag-V5 by immunofluorescence. The primary antibody directed againstthe V5 tag is a rabbit anti-V5 (polyclonal antibody, the company CovanceCat#PRB189P). The anti-rabbit secondary antibody is coupled to afluorophore of Alexa488 or Alexa594 type (Invitrogen). This detectionfinishes the validation of the cloning of the gene of interest.

A second cell culture series of dishes 60 mm in diameter, with the sametransfecting agent (Jet-PEI), makes it possible to produce a largeramount of cells. Two days after the transfection of the constructcontaining the gene of interest, the cells are recovered in thefollowing way:

The culture medium is removed.

The cells are rinsed with 1 ml of PBS which is immediately suctionedoff.

The cells are again rinsed with 1 ml of PBS.

They are left for 10 minutes at ambient temperature (AT).

All of the subsequent procedure is advantageously carried out in ice.

With a micropipette (P1000+filter tips), the cells are detached byrepeated and careful projection of the PBS contained the dish.

The resuspended cells are transferred into a 1.5 ml centrifugation tube(eppendorf type).

The cells are centrifuged for 5 minutes at 14K RPM (revolutions perminute) and at 4° C. in a bench centrifuge.

The supernatant is removed.

The cells are resuspended in 50 μl of solubilization buffer prepared,for example, as follows:

40% glycerol, 2% CHAPS, 140 mM NaCl, 2 mM EDTA, 20 mM Tris, pH8.8, inPBS to which an anti-protease mix of “Complete” type (from the companyRoche) has been added.

The cells are solubilized in this buffer by pipetting up and downapproximately 50 times.

Still in the microfuge tube, the solubilisate is left in ice for onehour.

A centrifugation is carried out for 10 minutes at 2500 RPM (revolutionsper minute), and at 4° C.

The supernatant is transferred into a clean tube, DNA loading buffer isadded, and the mixture is stored at −80° C.

Once the collection of several solubilisates of various cultures and ofvarious expressed proteins has been established, these soluble proteinsare distributed into conical-bottom 96-well plates, advantageously usingthe 24 central wells.

While maintaining these plates on ice, a fraction of each solubilisateis sampled using a multiwell replicator and a footprint is produced intriplicate on a nitrocellulose membrane (Hybond-C Exra Cat#RPM303C,Amersham).

This membrane constitutes the support for the 24 solubilisates to betested with the sample from a patient. An identical footprint isproduced in order to verify the correct expression of the V5-taggedproteins in each solubilisate.

On the day of the test, the footprinted membranes are cut to the sizelimit of the 24 wells. (One corner is notched in order to orient themembrane).

This membrane is rolled up and introduced into a 5 ml hemolysis tube.

It is ensured that the entire surface area of the membrane is uniformlydistributed inside the tube.

This membrane is incubated for 15 minutes with 2 ml of blocking solution(PBS, 5% milk, 0.2% NP40).

This blocking medium is replaced with 750 μl to 1 ml of antibodysolution at 1/20^(th) (50 μl of CSF in 1 ml of blocking solution).Everything is incubated overnight at ambient temperature on a rotarymachine.

The incubation medium is recovered and frozen for a second use.

Each membrane is rinsed three times in the hemolysis tubes with therinsing solution (PBS, 0.1% NP40).

Incubation is carried out for 2 hours at ambient temperature with thesecondary antibody (diluted to 1/5000^(th) in the blocking solution(PBS, 5% milk, 0.2% NP40)). The secondary antibody is coupled to HRP(HorseRadish Peroxidase) (anti-human and anti-rabbit from JacksonImmunoresearch).

Rinsing is carried out three times with the rinsing solution (PBS, 0.1%NP40) and once with PBS alone.

The blots are developed with 500 μl of ECL (Enhanced chemiluminescence)(Pierce) in the developing machine (FujiFilm Fluorescent Image AnalyzerFLA-3000).

Example 2 Validation of the Efficiency of the Protein Chip

In the present example, samples (of CSF type) originating from a cohortof patients known to have antibodies directed against the NMDA receptorsubunit NR1 (initially detected using an immunocytology method) and alsosamples originating from individuals not suffering from an autoimmunedisease are used to validate the efficiency of the protein chipaccording to the invention.

The invention was validated by means of a blind test carried out onvarious samples originating from 17 patients known to contain anti-NR1antibodies (in a population consisting of 26 healthy individuals).

According to the protocol described above, the membrane proteins ofcells expressing the NR1 receptor are solubilized and deposited on anitrocellulose membrane. Each membrane has four deposits of the proteinof interest corresponding to four decreasing dilutions of the samesolubilized membrane protein preparation resulting from cellsoverexpressing the NR1 subunit, one control deposit corresponding tosolubilized membrane proteins from cells expressing the empty vector,and one control deposit corresponding to a solution of human serum froma healthy subject (see FIG. 1). After incubation and detection asdescribed above, the results show that, of the 17 afflicted patients,the test recognized 15 thereof and therefore has an efficiency of 88%.The specificity for the negative patients is 100%. The two falsepositives recognized by the protein chip according to the inventioncorrespond to two candidates who had been detected as positive and thendiscarded in a second screening of a first test carried out on cells.

Example 3 Screening Using the Protein Chip

In the present example, the invention was tested on a cohort of 50patients suffering from neurological disorders of unknown origin (andwhich were potentially autoimmune) for whom the inventors sought to knowwhether they had antibodies directed against one or more proteins among40 proteins deposited on a nitrocellulose membrane.

In this example, each plate (FIG. 2) possesses 20 samples of solubilizedmembrane proteins from cells individually overexpressing 20 proteins ofinterest (codified here in A15, F28, F31, etc.) and also two samplescorresponding to a dilution of human serum from a healthy subject.

The deposits on nitrocellulose membrane were carried out according tothe protocol described above (with a replicator) in duplicate and whilecombining the deposits of the two plates (plate 1 at the bottom, plate 2at the top) on the same membrane (see FIG. 2).

The results show that, among the 50 patients tested, many patients werenegative and therefore did not have antibodies directed against one ofthe 40 proteins deposited, a small number had antibodies against all theproteins deposited, and one patient had antibodies directed specificallyagainst one protein (H7).

After comparison of these results with the clinical picture of thecohort of 50 patients, it appears that the non-specific response of thepatients having antibodies directed against all the proteins is partlyexplained by the degree of advancement and of severity of their disease,since most of them died even before the test was set up (massive andnon-specific immune response). On the other hand, the sole resultregarding the H7 protein is the perfect example of the possibleidentification, using the chip according to the invention, of theassociation (demonstrated for the first time) that exists between aparticular channel (protein identified in the present example as the H7protein) and a neurological disease.

1-19. (canceled)
 20. A protein chip for detecting autoantibodies in a biological sample, said chip comprising at least one solubilized membrane protein of interest expressed in the cells of the nervous system of an animal, involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders, said membrane protein being optionally associated with one or more auxiliary subunits, and said chip being in the form of a support for detection of the complexes formed between i) autoantibodies and sequential epitopes and ii) autoantibodies and conformational epitopes, said sequential and conformational epitopes being specific for said membrane protein, optionally associated with its auxiliary subunit(s).
 21. The chip according to claim 20, characterized in that said at least one solubilized membrane protein of interest is chosen from an ion channel protein, a transporter protein, and a membrane receptor protein which regulates the activity of an ion protein channel or of a transporter protein.
 22. The chip according to claim 20, characterized in that said at least one membrane protein is expressed in the cells of the nervous system of a human being.
 23. The chip according to claim 20, characterized in that the encephalopathy associated with neuronal excitability disorders is selected from post-infectious encephalitis, anti-NMDAR encephalitis, Morvan's syndrome, Devic's disease, opsoclonus myoclonus in children, limbic encephalitis, Rasmussen encephalitis, encephalomyelitis, acquired epileptic syndrome and paraneoplastic neurological syndrome (PNS).
 24. The chip according to claim 20, characterized in that said at least one membrane protein consists of an amino acid sequence chosen from SEQ ID NO: 1 to SEQ ID NO:311.
 25. The chip according to claim 20, characterized in that the membrane protein of interest is chosen from a GABA receptor; a glutamate receptor; aquaporin 2 (SEQ ID NO: 307); aquaporin 4 (SEQ ID NO: 309); a potassium channel; a calcium channel; an acetylcholine receptor, a protein associated with voltage gated potassium channels; a glycine receptor; connexin 32 (SEQ ID NO: 310) and connexin 43 (SEQ ID NO: 311).
 26. The chip according to claim 25, characterized in that the membrane protein of interest is an N-methyl-D-aspartate (NMDA)-activated glutamate receptor.
 27. The chip according to claim 26, wherein the NMDA-activated glutamate receptor is the NMDAR1 receptor (SEQ ID NO: 62).
 28. The chip according to claim 20, characterized in that it comprises: a) at least 100 different membrane proteins of interest, b) between approximately 200 and approximately 400 different proteins of interest; c) approximately 250; or d) approximately 300 different proteins of interest, wherein each protein of interest is optionally associated with one or more auxiliary subunits.
 29. The chip according to claim 20, characterized in that said membrane protein(s) of interest is (are) labelled with the same peptide sequence recognized by a detection antibody.
 30. The chip according to claim 20, characterized in that it also comprises at least one positive control and one negative control.
 31. The chip according to claim 20, characterized in that it comprises a support capable of binding solubilized membrane proteins of interest, selected from a membrane; glass; and plastic.
 32. The chip according to claim 31, wherein the membrane is a nitrocellulose or nylon membrane.
 33. The chip according to claim 20, in which the biological sample originates from a mammal and is chosen from blood, serum, plasma, cerebrospinal fluid (CSF), inner ear endolymph, inner ear perilymph, and a subfraction thereof.
 34. The chip according to claim 33, wherein the mammal is a human.
 35. A method for preparing a chip according to claim 20, comprising the following steps: cloning the cDNA(s) of interest, expressing the membrane protein(s) expressed in the cells of the nervous system of an animal, which is (are) involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders, and encoded by said cDNA(s), in cells in culture, solubilizing the proteins expressed using a non-denaturing detergent which allows the solubilizing of said membrane proteins while at the same time preserving their conformation and their ability to bind to a support, and depositing said solubilized membrane proteins on a support, thereby obtaining the chip of interest.
 36. The method according to claim 35, characterized in that the detergent is a detergent chosen from triton X-100, triton X-114, Nonidet P-40, CHAPS, sodium cholate, sodium deoxycholate, digitonin, sarkosyl NL30 and octylglucoside.
 37. A chip obtainable by means of a method according to claim
 35. 38. A kit comprising a chip according to claim 20, and, optionally, one or more reagents chosen from a buffer for blocking the non-specific sites, a buffer allowing the association between antibodies and antigens, a washing buffer, a secondary antibody, a product for detecting said secondary antibody, and instructions for use. 